Biflavanoids and derivatives thereof as antiviral agents

ABSTRACT

Substantially purified antiviral biflavanoids robustaflavone, hinokiflavone, amentoflavone, agathisflavone, volkensiflavone, morelloflavone, rhusflavanone, succedaneaflavanone, GB-1a, and GB-2a are provided. Antiviral biflavanoid derivatives and salt forms thereof, e.g., robustaflavone tetrasulfate potassium salt, and methods for preparing the same are also disclosed. Pharmaceutical compositions which include the antiviral biflavanoids, derivatives or salts thereof are also provided alone or in combination with at least one antiviral agent such as 3TC. Also disclosed is an improved method for obtaining substantially pure robustaflavone from plant material. The biflavanoid compounds, derivatives or salts thereof of the invention may be used in a method for treating and/or preventing viral infections caused by viral agents such as influenza, e.g., influenza A and B; hepatitis, e.g., hepatitis B; human immunodeficiency virus, e.g., HIV-1; Herpes viruses (HSV-1 and HSV-2); Varicella Zoster virus (VZV); and measles. For instance, semi-synthetic hexa-O-acetate and hexa-O-methyl ether derivatives of robustaflavone have been found to be effective in a method for treating or preventing hepatitis B viral infections. Compositions which include these robustaflavone derivatives along with methods for preparing and using the same are also provided. These compositions may be used alone or in combination with at least one antiviral agent such as 3TC.

CROSS-REFERENCE

[0001] This application is a continuation-in-part of U.S. Ser. No.08/842,625, filed Apr. 15, 1997 which is continuation-in-part of U.S.Ser. No. 08/668,284, filed Jun. 21, 1996, which in turn is acontinuation-in-part of provisional application No. 60/000465, filedJun. 23, 1995.

[0002] This invention was supported in part by NIAID N01-AI45195 and NIHgrant No. 1R43AI40745-01. The U.S. government has certain rights to thisinvention.

FIELD OF THE INVENTION

[0003] The present invention relates to substantially pure antiviralbiflavanoids, e.g., robustaflavone, biflavanoid derivatives and saltsthereof such as esters, ethers, amines, sulfates, ethylene oxideadducts, and acid salts, and pharmaceutical compositions containing thesame. Representative examples include hexa-O-acetate and hexa-O-methylether derivatives of robustaflavone and robustaflavone tetrasodium salt.The present invention also relates to methods for extractingsubstantially pure robustaflavone from plant material. The presentinvention also relates to a method for preventing and/or treating viralinfections such as hepatitis B, influenza A and B, and HIV which employrobustaflavone or derivatives thereof alone or in combination with atleast one antiviral agent such as 3TC.

BACKGROUND OF THE INVENTION

[0004] Viruses, an important etiologic agent in infectious disease inhumans and other mammals, are a diverse group of infectious agents thatdiffer greatly in size, shape, chemical composition, host range, andeffects on hosts. After several decades of study, only a limited numberof antiviral agents are available for the treatment and/or prevention ofdiseases caused by viruses such as hepatitis B, influenza A and B andHIV. Because of their toxic effects on a host, many antiviral agents arelimited to topical applications. Accordingly, there is a need for safeand effective antiviral agents with a wide-spectrum of anti-viralactivity with reduced toxicity to the host.

[0005] Since the identification of the human immunodeficiency virus(HIV) as the causative agent of AIDS,^(36,46) the search for safe andeffective treatments for HIV infection has become a major focus for drugdiscovery groups around the world. Investigations into the molecularprocesses of HIV have identified a number of macromolecular targets fordrug design, such as HIV-1 reverse transcriptase (HIV-RT), protease andintegrase enzymes, and regulatory proteins (e.g., TAT and REV). Othertargets are enzymes which aid in virus attachment and fusion. HIV-RT isan essential enzyme in the life cycle of HIV, which catalyzes thetranscription of HIV-encoded single-stranded RNA into double-strandedDNA. Furthermore, the RNA-dependent DNA polymerase function of HIV-RTdoes not have an analogous process in mammalian metabolism, and thus isa suitable target for a chemotherapeutic agent.

[0006] The hepatitis B virus (HBV) infects people of all ages. It is oneof the fastest-spreading sexually transmitted diseases, and also can betransmitted by sharing needles or by behavior in which a person's mucusmembranes are exposed to an infected person's blood, semen, vaginalsecretions, or saliva. While the initial sickness is rarely fatal, tenpercent of the people who contract hepatitis are infected for life andrun a high risk of developing serious, long-term liver diseases, such ascirrhosis of the liver and liver cancer, which can cause seriouscomplications or death.¹ The World Health Organization lists HBV as theninth leading cause of death. It is estimated that about 300 millionpersons are chronically infected with HBV worldwide, with over 1 millionof those in the United States. The Center for Disease Control estimatesthat over 300,000 new cases of acute HBV infection occurs in the UnitedStates each year, resulting in 4,000 deaths due to cirrhosis and 1,000due to hepatocellular carcinoma.² The highest rates of HBV infectionsoccur in Southeast Asia, South Pacific Islands, Sub-Saharan Africa,Alaska, Amazon, Bahai, Haiti, and the Dominican Republic, whereapproximately 20% of the population is chronically infected.³

[0007] Hepatitis B virus (HBV) infection is currently the most importantchronic virus infection, but no safe and effective therapy is availableat present. The major therapeutic option for carriers of HBV is alphainterferon, which can control active virus replication. However, even inthe most successful studies, the response rate in carefully selectedpatient groups has rarely exceeded 40%.^(5,6) One of the reasons citedfor interferon failure is the persistence of viral supercoiled DNA inthe liver.⁷

[0008] Recently, lamivudine (3TC) has provided encouraging resultsagainst both HBV and HIV in human clinical trials. Lamivudine isapproved for treatment of HIV infection, and is currently beingevaluated as a treatment for HBV. A recent study of 40HIV-HBV-coinfected patients showed dramatic decreases in the level ofHBV replication following treatment with 3TC over a 12 month period,with no observable adverse effects (84). Another agent, famciclovir, anorally active derivative of the acyclic guanine derivative penciclovir(85), is approved for treatment of herpes zoster and acute recurrentgenital herpes, and has also shown promising results against HBVinfection in clinical trials, again with little observable toxicity(86). It is hoped that these agents will eventually provide promisingtreatment options for HBV infection.

[0009] Unfortunately, monotherapy of viral infections often results inselection for mutant viral strains having resistance to the antiviraldrug being used. Indeed, clinical isolates of mutant HBV strains havebeen identified having resistance to 3TC following treatment with thatagent (87,88). It is plausible that combination therapy of HBV wouldprovide an enhanced antiviral response, while reducing the danger ofresistance selection. Combination of agents has been shown to besuperior in treatment of HIV relative to monotherapy (89). Thus, theidentification of compounds which inhibit HBV, particularly compoundshaving structures other than that of the nuceloside analogues, is ofcritical importance in the search for effective anti-HBV regimens.

[0010] Influenza is a viral infection marked by fever, chills, and ageneralized feeling of weakness and pain in the muscle, together withvarying signs of soreness in the respiratory tract, head, and abdomen.Influenza is caused by several types of myxoviruses, categorized asgroups A, B, and C₄. These influenza viruses generally lead to similarsymptoms but are completely unrelated antigenically, so that infectionwith one type confers no immunity against the other. Influenza tends tooccur in wavelike epidemics throughout the world; influenza A tends toappear in cycles of two to three years and influenza B in cycles of fourto five years. Influenza is one of the few common infectious diseasesthat are poorly controlled by modem medicine. Its annual epidemics areoccasionally punctuated by devastating pandemics. For example, theinfluenza pandemic of 1918, which killed over 20 million people andaffected perhaps 100 times that number, was the most lethal plague everrecorded. Since that time, there have been two other pandemics of lesserseverity, the so-called Asian flu of 1957 and the Hong Kong flu of 1968.All of these pandemics were characterized by the appearance of a newstrain of influenza virus to which the human population had littleresistance and against which previously existing influenza virusvaccines were ineffective. Moreover, between pandemics, influenza virusundergoes a gradual antigenic variation that degrades the level ofimmunological resistance against renewed infection.⁴

[0011] Anti-influenza vaccines, containing killed strains of types A andB virus currently in circulation, are available, but have only a 60 to70% success rate in preventing infection. The standard influenza vaccinehas to be redesigned each year to counter new variants of the virus. Inaddition, any immunity provided is short-lived. The only drugs currentlyeffective in the prevention and treatment of influenza are amantadinehydrochloride and rimantadine hydrochloride.¹¹⁻¹³ While the clinical useof amantadine has been limited by the excess rate of CNS side effects,rimantadine is more active against influenza A both in animals and humanbeings, with fewer side effects.^(14,15) It is the drug of choice forthe chemoprophylaxis of influenza A.^(13,16,17) However, the clinicalusefulness of both drugs is limited by their effectiveness against onlyinfluenza A viruses, by the uncertain therapeutic efficacy in severeinfluenza, and by the recent findings of recovery of drug-resistantstrains in some treated patients.¹⁸⁻²² Ribavirin has been reported to betherapeutically active, but it remains in the investigational stage ofdevelopment.^(23,24)

[0012] In influenza, amantadine and rimantadine have been shown to bemoderately effective against only influenza A viruses; with amantadinehaving excessive side effects. Recently, strains of influenza Aresistant to amantadine and rimantadine have been isolated. Accordingly,there is a need for new types of therapeutic antiviral agents againstboth influenza A and influenza B, as well as against HBV and HIV.

SUMMARY OF THE INVENTION

[0013] The present invention relates to substantially purified antiviralbiflavanoids, derivatives and salts thereof and pharmaceuticalcompositions containing the same; improved methods for extractingsubstantially pure robustaflavone from plant material; methods forpreparing derivatives and salts from antiviral biflavanoids; and methodsfor treating and/or preventing viral infections using the antiviralbiflavanoids, derivatives and salts thereof.

[0014] The present invention provides substantially purifiedbiflavanoids comprising robustaflavone, hinokiflavone, amentoflavone,agathisflavone, morelloflavone, volkensiflavone, rhusflavanone,succedaneaflavanone, GB-1a, and GB-2a and pharmaceutical compositionscontaining the same. Scheme I illustrates the chemical structures ofthese biflavanoids. The biflavanoids of the invention, extractable fromplant materials derived from a variety of natural sources such as Rhussuccedanea and Garcinia multiflora, were found to be effective ininhibiting viral activity and may be used in a method for treatingand/or preventing a broad range of viral infections such as Influenza Aand B, hepatitis B and HIV-1, HSV-1, HSV-2, VZV, and measles. It hasbeen discovered that robustaflavone effectively inhibits activity ofinfluenza A and B viruses, hepatitis B, HIV-1, HSV-1 and HSV-2.Hinokiflavone and morelloflavone exhibited similar activity againstvarious strains of HIV-1.

[0015] Anti-viral biflavanoid derivatives and salts and pharmaceuticalcompositions containing the same are also contemplated by the invention.Representative derivatives include ethers, e.g., methyl ethers, esters,amines, ethylene oxide adducts, and polymers such as trimers andtetramers of apigenin. Representative salts include sulfates and acidsalts. Methods for preparing these derivatives and salts are alsoprovided. It has been discovered for instance that salts ofrobustaflavone, e.g., robustaflavone tetrasulfate potassium salt androbustaflavone tetrasodium salt, effectively inhibit hepatitis Bactivity. Furthermore, robustaflavone hexa-O-acetate and hexa-O-methylether derivatives of robustaflavone were found to be not only potentinhibitors of HBV replication but with greatly reduced cytotoxicity.Scheme I illustrates several examples of biflavanoid derivatives.

[0016] Improved methods for extracting robustaflavone from plantmaterial are also provided. According to one method, a substantiallypure robustaflavone in greater yields can be obtained through the use ofa particular solvent mixture comprising toluene/ethanol/pyridine. Theimproved extraction method eliminates the use of benzene and requiressmaller volumes of pyridine from the prior reported methods.

[0017] A second improved method for purification of robustaflavone isalso provided which involves acetylation of the extracted pigment toproduce acetates of the pigments. Robustaflavone acetate is thenpurified by recrystallization and converted to robustaflavone byhydrolysis. This method eliminates the use of pyridine in columnchromatography and is ideal for large scale extraction ofrobustaflavone.

[0018] Finally, a method for treating and/or preventing viral infectionsusing antiviral biflavanoids alone or in combination with one or moreother antiviral agents is described. Representative viral infectionsinclude influenza A and B viruses, hepatitis B and humanimmunodeficiency virus (HIV-1), HSV-1, HSV-2, VZV, and measles.

[0019] Accordingly, it is an object of the invention to providesubstantially purified antiviral biflavanoids robustaflavone,hinokiflavone, amentoflavone, agathisflavone, morelloflavone,rhusflavanone, succedaneaflavanone, GB-1a, and GB-2a.

[0020] It is another object of the invention to provide antiviralderivatives and salt forms of biflavanoids robustaflavone,hinokiflavone, amentoflavone, agathisflavone, morelloflavone,volkensiflavone, rhusflavanone, succedaneaflavanone, GB-1a, and GB-2a aswell as method of preparation thereof. A representative example of anantiviral biflavanoid derivatives include robustaflavone tetrasulfatepotassium salt, robustaflavone tetrasodium salt, robustaflavonehexa-O-acetate, and robustaflavone hexa-O-methyl ether.

[0021] It is yet another object of the invention to providepharmaceutical compositions which include at least one antiviralbiflavanoids such as robustaflavone, hinokiflavone, amentoflavone,agathisflavone, morelloflavone, volkensiflavone, rhusflavanone,succedaneaflavanone, GB-1a, GB-2a, derivatives or salts thereof.Pharmaceutical compositions including an effective amount of at leastone antiviral biflavanoid in combination with at least one otherantiviral agent, e.g. robustaflavone with penciclovar or lamivudine(3TC), for use in combination antiviral therapy are also contemplated.

[0022] It is a further object of the invention to provide an improvedmethod for obtaining substantially pure robustaflavone and in greateryields than prior procedures.

[0023] It is yet a further object of the invention to provide a methodfor treating and/or preventing viral infections which comprisesadministering an antiviral effective amount of a biflavanoid.Representative viral infections are caused by viral agents such asinfluenza, e.g., influenza A and B; hepatitis, e.g., hepatitis B; humanimmunodeficiency virus, e.g., HIV-1; HSV-1, HSV-2, VZV, and measles.

[0024] These and other objects of the invention will become apparent inlight of the detailed description below.

DESCRIPTION OF THE DRAWINGS

[0025]FIG. 1 illustrates the effect of treatment with robustaflavone inDMSO on mean arterial oxygen saturation (mean SaO₂ (%)) in Influenza Avirus-infected mice as described in Example 11.

[0026]FIG. 2 illustrates the effect of treatment with robustaflavone inDMSO on mean lung scores in Influenza A virus-infected mice as describedin Example 11.

[0027]FIG. 3 illustrates the effect of treatment with robustaflavone inDMSO on mean lung weights in Influenza A virus-infected mice asdescribed in Example 11.

[0028]FIG. 4 illustrates the effect of treatment with robustaflavone inDMSO on mean virus titers in Influenza A virus-infected mice asdescribed in Example 11.

[0029]FIG. 5 illustrates the effect of treatment with robustaflavone inCMC on mean arterial oxygen saturation (mean SaO₂ (%)) in Influenza Avirus-infected mice as described in Example 11.

[0030]FIG. 6 illustrates the effect of treatment with robustaflavone inCMC on mean lung scores in Influenza A virus-infected mice as describedin Example 11.

[0031]FIG. 7 illustrates the effect of treatment with robustaflavone inCMC on mean lung weights in Influenza A virus-infected mice as describedin Example 11.

[0032]FIG. 8 illustrates the effect of treatment with robustaflavone inCMC on mean virus titers in Influenza A virus-infected mice as describedin Example 11.

DETAILED DESCRIPTION OF THE INVENTION

[0033] All references and patents cited herein are hereby incorporatedby reference in their entirety.

[0034] In one embodiment of the invention, substantially purebiflavanoids robustaflavone, hinokiflavone, amentoflavone,agathisflavone, morelloflavone, volkensiflavone, rhusflavanone,succedaneaflavanone, GB-1a, and GB-2a, derivatives and salts of thebiflavanoids, and pharmaceutical compositions containing the same aredisclosed. Methods for extracting and isolating the biflavanoids werepreviously reported.^(28,37,39,40,53-55) Moreover, methods for preparingderivatives such as the acetate^(37,38) and methyl ethers^(39,40) forseveral of these biflavanoids are also reported. Representative methodsfor preparing biflavanoid derivatives are illustrated in the examplesbelow. Applicants have determined that these biflavanoids, especiallyrobustaflavone, were surprisingly effective in inhibiting one or moreactivities of viruses such as Influenza A and B, hepatitis B and HIV-1,HSV-1, HSV-2, VZV, and measles.

[0035] Approximately 100 biflavanoids have been isolated to date, sincethe first biflavanoid, a biflavone, was isolated in 1929 by Furukawafrom ginkgo biloba L. as a yellow pigment.^(44,45,61) Biologicalactivities of several biflavanoids, such as ginkgetin, have beenreported. For instance, peripheral vasodilatation, anti-bradykinin, andanti-spasmogenic activities have been observed.^(48,62) Garcinikolinstimulates RNA synthesis in rat hepatocyte suspensions.⁵⁷ Also,agathisflavone, kolaviron, GB-1 and GB-2 have hepatoprotectiveactivity.^(33,49) Hinokiflavone, kayaflavone, bilobetin, lophirone A,lophiraic acid, and sotetusflavone demonstrate inhibitory action on thegenome expression of the Epstein-Barr virus (EBV).^(51,52,60) GB-1exhibits molluscicidal activity,⁶⁵ while daphnodorin A, daphnodorin B,and daphnodorin D possess antimicrobial activity.³⁴ Hinokiflavoneexhibits cytotoxicity against tissue cultured cells of human mouthepidermoid carcinoma (KB).⁵⁶ Amentoflavone and morelloflavone exhibit aninhibitory effect on lipid peroxidation,^(41,59,66) and kolavironproduced hypoglycemic effects.⁵⁰ None of these references, however,disclose or suggest that robustaflavone, hinokiflavone, morelloflavone,amentoflavone, agathisflavone, volkensiflavone, rhusflavanone,succedaneaflavanone, GB-1a and GB-2a, especially robustaflavone and itstetrasulfate potassium salt, have an inhibitory effect against at leastone of influenza, e.g., influenza A and B; hepatitis, e.g., hepatitis B;human immunodeficiency virus, e.g., HIV-1; HSV-1, HSV-2, VZV, andmeasles.

[0036] In another embodiment of the invention, an improved method forextracting substantially pure robustaflavone from natural sources isalso provided. Robustaflavone, 1, a naturally occurring biflavanoid, waspreviously isolated, purified, and identified from the seed-kemels ofRhus succedanea. ²⁵ Other sources of robustaflavone include: seed kernelof Rhus succedanea L.;²⁵ leaves of Selaginella lepidophylla; ²⁷ leavesof Anacardium occidentale; ²⁸ leaves and branches of Podocarpusneriifolius D. Doa;²⁹ Selaginella denticulata; ³⁰ and Selaginellawilldenowii. ³¹

[0037] The drupes of wax-tree, Rhus succedanea L (Anacardiaceae), are ofgreat economic importance in that they yield Japan wax. Earlier work onthis species has shown the presence of fustin and fisetin in the wood,rhoifolin in leaves, japanic acid in the wax, and ellagic acid, fattyacids, and flavanoids in the seed kernels. Further studies of thepigment in the seed kernels of wax-tree led to the isolation of eightbiflavanoids, four of which were new. Concentration of the ethanolextract of the seed kernels yielded, successively, fractions of ellagicacid, pigment A (hinokiflavone and robustaflavone) and pigment B(amentoflavone). Further concentrations gave a crude yellow pigment Cwhich, when subjected to silica gel column chromatography, affordedfractions C_(I) (rhusflavanone, succedaneaflavanone andneorhusfiavanone), C_(II) (rhusflavone), and C_(III) (agathisflavone).

[0038] A prior method for extracting and isolating substantially purerobustaflavone from plant material was reported.⁵⁵ This method, however,used large quantities of benzene and pyridine which is undesirable foruse in large scale extractions and produced mediocre yields ofrobustaflavone. The Applicants discovered improved extraction methodswhich eliminates benzene, greatly reduces or eliminates the amount ofpyridine used and produces at least double the quantities ofsubstantially pure robustaflavone compared to the prior method. In onemethod of the invention, solvent mixture comprisingtoluene/ethanol/formic acid at a volume ratio ranging about10-30:2-10:1, preferably about 20:5:1, was found to be useful as adeveloping solvent mixture for the dry column procedure. This particularsolvent mixture was found to be especially useful in large scaleextractions.

[0039] In a second method for purification of robustaflavone, extractedpigment is acetylated followed by purification and hydrolysis of therobustaflavone acetate to produce pure robustaflavone. This methodeliminates the use of pyridine in column chromatography and is ideal forlarge scale isolation of purified robustaflavone. According to thesecond method, pigment A was converted to the acetate with an acylatingagent in solution. Suitable, but non-limiting, acylating agents includeacetic anhydride and acetyl chloride. The preferred acylating agent isacetic anhydride. In general, excess amounts of acylating agent relativeto pigment A are used. Suitable, but non-limiting, solvents for use inthis second method include any suitable solvent generally used foracetylation reactions including pyridine and triethylamine. Inpracticing this invention, pyridine is the preferred solvent.

[0040] The acetylation reaction is generally performed at roomtemperature until completion is reached as ascertained by the usualorganic chemical methods such as thin layer chromatography or Gas liquidchromatography. Thereafter, the crude acetate is precipitated out inwater and collected by filtration. The crude precipitate is then washedwith water and recrystallized out from a suitable solvent or solventmixture. In practicing this invention, robustaflavone acetate ispreferrably recrystallized from a 9:1 solvent mixture of methylenechloride and ethyl acetate. If desired, additional acetate may berecovered from the mother liquor by column chromatography. The purifiedrobustaflavone acetate is then hydrolyzed with excess aqueous alkalinesolution such as 2M aqueous sodium hydroxide solution. In practicingthis invention, robustaflavone acetate hydrolysis mixture is preferablyto a temperature ranging between about 30 to about 100° C., preferablyaround 60° C., for a time sufficient to hydrolyze off the acetategroups. The alkaline reaction mixture is then cooled in an ice bath andacidified to about pH 3.0 with excess amounts of aqueous acid solution,e.g., 3N hydrochloric acid, to precipitate out robustaflavone as ayellow solid.

[0041] Examples of extraction via the improved extraction methods of theinvention are illustrated in the examples below.

[0042] In yet another embodiment of the invention, a method is providedfor treating and/or preventing viral infections in mammals comprisingadministering an antivirally effective amount of a biflavanoid suchrobustaflavone, hinokiflavone, amentoflavone, agathisflavone,morelloflavone, volkensiflavone, rhusflavanone, succedaneaflavanone,GB-1a, and GB-2a. In practicing this invention, administration ofrobustaflavone or derivatives thereof is preferred. Examples of mammalsinclude humans, primates, bovines, ovines, porcines, felines, canines,etc. Examples of viruses may include, but not be limited to, HIV-1,HIV-2, herpes simplex virus (type 1 and 2) (HSV-1 and 2), varicellazoster virus (VZV), cytomegalovirus (CMV), papilloma virus, HTLV-1,HTLV-2, feline leukemia virus (FLV), avian sarcoma viruses such as roussarcoma virus (RSV), hepatitis types A-E, equine infections, influenzavirus, arboviruses, measles, mumps and rubella viruses. More preferablythe compounds of the present invention will be used to treat a humaninfected with hepatitis and/or influenza virus. Preferably the compoundsof the present invention will also be used to treat a human exposed orinfected (i.e., in need of such treatment) with the humanirnmunodeficiency virus, either prophylactically or therapeutically.

[0043] Antiviral biflavanoids and derivatives thereof may be formulatedas a solution of lyophilized powders for parenteral administration.Powders may be reconstituted by addition of a suitable diluent or otherpharmaceutically acceptable carrier prior to use. The liquid formulationis generally a buffered, isotonic, aqueous solution. Examples ofsuitable diluents are normal isotonic saline solution, standard 5%dextrose in water or in buffered sodium or ammonium acetate solution.Such formulation is especially suitable for parenteral administration,but may also be used for oral administration. It may be desirable to addexcipients such as polyvinylpyrrolidone, gelatin, hydroxy cellulose,acacia, polyethylene glycol, mannitol, sodium choride or sodium citrate.

[0044] Alternatively, the compounds of the present invention may beencapsulated, tableted or prepared in an emulsion (oil-in-water orwater-in-oil) syrup for oral administration. Pharmaceutically acceptablesolids or liquid carriers, which are generally known in thepharmaceutical formulary arts, may be added to enhance or stabilize thecomposition, or to facilitate preparation of the composition. Solidcarriers include starch (corn or potato), lactose, calcium sulfatedihydrate, terra alba, croscarmellose sodium, magnesium stearate orstearic acid. talc, pectin, acacia, agar, gelatin, maltodextrins andmicrocrystalline cellulose, or collodial silicon dioxide. Liquidcarriers include syrup, peanut oil, olive oil, corn oil, sesame oil,saline and water. The carrier may also include a sustained releasematerial such as glyceryl monostearate or glyceryl distearate, alone orwith a wax. The amount of solid carrier varies but, preferably, will bebetween about 10 mg to about 1 g per dosage unit.

[0045] The dosage ranges for administration of biflavanoids orderivatives thereof are those which produce the desired affect wherebysymptoms of infection are ameliorated. For example, as used herein, apharmaceutically effective amount for influenza or hepatitis infectionrefers to the amount administered so as to maintain an amount whichsuppresses or inhibits circulating virus throughout the period duringwhich infection is evidenced such as by presence of anti-viralantibodies, presence of culturable virus and presence of viral antigenin patient sera. The presence of anti-viral antibodies can be determinedthrough use of standard ELISA or Western blot assays for example. Thedosage will generally vary with age, extent of the infection, the bodyweight and counterindications, if any, for example, immune tolerance.The dosage will also be determined by the existence of any adverse sideeffects that may accompany the compounds. It is always desirable,whenever possible, to keep adverse side effects to a minimum.

[0046] One skilled in the art can easily determine the appropriatedosage, schedule, and method of administration for the exact formulationof the composition being used in order to achieve the desired effectiveconcentration in the individual patient. However, the dosage can varyfrom between about 0.001 mg/kg/day to about 150 mg/kg/day, butpreferably between about 1 to about 50 mg/kg/day.

[0047] The pharmaceutical composition may contain other pharmaceuticalsin conjunction with biflavanoids and derivatives thereof to treat(therapeutically or prophylactically) antiviral infections. For example,other pharmaceuticals may include, but are not limited to, otherantiviral compounds (e.g., AZT, ddC, ddI, D4T, 3TC, acyclovir,gancyclovir, fluorinated nucleosides and nonnucleoside analog compoundssuch as TIBO derivatives, nevirapine, saquinavir, a-interfon andrecombinant CD4), immunostimulants (e.g., various interleukins andcytokines), immunomodulators and antibiotics (e.g., antibacterial,antifungal, anti-pneumocysitis agents).

[0048] The following examples are illustrative and do not serve to limitthe scope of the invention as claimed. In these examples, elevenbiflavanoids, amentoflavone (1), agathisflavone (2), robustaflavone (3),hinokiflavone (4), volkensiflavone (5), morelloflavone (7),rhusflavanone (9), succedaneaflavanone (11), GB-1a (13), GB-1a7″-O-b-glucoside (15), and GB-2a (16), isolated from Rhus succedanea andGarcinia multiflora, and their methyl ethers, acetate and sulfatepotassium salt, volkensiflavone hexamethyl ether (6), morelloflavoneheptamethyl ether (8), rhusflavanone hexaacetate (10)succedaneaflavanone hexaacetate (12), GB-1a hexamethyl ether (14) androbustaflavone tetrasulfate potassium salt were evaluated for theirantiviral activities. The inhibitory activities against HIV-1 RT andvarious viruses including herpes viruses (HSV-1, HSV-2, HCMV, and VZV),and respiratory viruses (influenza A, influenza B, RSV, parainfluenza 3,adenovirus 5, and measles) were investigated.

EXAMPLE 1 Extraction and Isolation of Biflavanoids Isolation ofCompounds

[0049] Compounds tested were isolated from the seed kernels of Rhussuccedanea obtained from Fukkuoka, Japan, and also from the heartwood ofGarcinia multiflora collected in Taiwan.

[0050] Amentoflavone (1),⁵³ agathisflavone (2),⁵⁴ robustaflavone (₃),⁵⁵hinokiflavone (4),⁵⁵ rhusflavanone (9),³⁷ and succedaneaflavanone (11)²⁸were isolated from Rhus succedanea. Rhusflavanone hexaacetate (10) andsuccedaneaflavanone hexaacetate (12) were prepared directly fromcompounds (9) and (11), respectively.^(37,38) Volkensiflavone (5),morelloflavone (7), GB-1a (13), GB-1a glucoside (15), and GB-2a (16)were isolated from Garcinia multiflora. ^(39,40) Volkensiflavonehexamethylether (6), morelloflavone hexamethylether (8), and GB-1ahexamethylether (14) were prepared from compounds (5), (7), and (13),respectively.^(39,40) Robustaflavone tetrasulfate potassium salt (17)was prepared from robustaflavone (3).

[0051] In this example, two procedures for isolating robustaflavone aredescribed. In the first procedure, robustaflavone was isolated by adry-column method using benzene/pyridine/formic acid (20:5:1) asdeveloping solvent, following an earlier reported procedure.²⁵ In orderto eliminate the use of benzene and large quantities of pyridine, animproved procedure was developed wherein benzene and pyridine arereplaced with other solvents. The solvent mixture oftoluene/ethanol/formic acid in the ratio of 20:5:1 was used as thedeveloping solvent in the dry-column procedure. Hinokiflavone was elutedcompletely from the dry-column and robustaflavone retained in thecolumn. A mixture of ethanol and pyridine in the ratio of 4:1 was thenused to elute robustaflavone from the column.

[0052] Extraction of Biflavanoids from Rhus succedanea. The seeds (16kg) of Rhus succedanea obtained from Fukuoka, Japan were coarselypowdered and defatted with benzene. The defatted seeds were exhaustivelyextracted with boiling 95% EtOH (150 L). The combined EtOH extracts wereconcentrated in vacuo. The yellow pigments obtained during theconcentration were filtered to yield crude pigment A (yield 0.2%) andpigment B (yield 0.2%). successively. Further concentration yieldedyellow pigment C (ca. 2%).

[0053] Isolation of Robustaflavone from Pigment A. One gram of pigment Adissolved in 10 mL of pyridine was mixed with 5 g of silica gel(Kiselgel nach Stahl Type 60 Merck) and evaporated in vacuo to removepyridine. The dried yellow powder obtained was packed on the top of asilica gel column (SiO₂100 g, 4×20 cm). The solvent mixture (400 mL) ofbenzene/pyridine/formic acid (40:10:2) was passed through the column.The column was sliced into seven bands (bands 1˜7 from top to bottom).Extraction of the yellow band 4 with EtOAc and subsequent concentrationof the extract yielded yellow crystals (200 mg), robustaflavone, whichwere recrystallized from pyridine-water, m.p. 350-352° C. (dec.). Mg-HCltest (orange red color), FeCl₃/EtOH test (brown color). IR cm⁻¹ (KBr):3300 (OH), 1655, 1645 (CO), 1610, 1570, 1510, 1505, 1485 (aromaticring), UVl_(max) (MeOH) nm (log ε): 255 (4.71), 275 (4.44), 300 (4.42),347 (4.49), l_(max) (NaOAc-MeOH) nm (log ε): 257 (4.66), 277 (4.48), 313(sh, 4.41), 378 (4.38), l_(max) (AlCl₃-MeOH) nm (log ε): 254 (4.80), 278(4.45), 300 (4.45), 352 (4.50), 388 (4.43); NMR (DMSO-d₆) (60 MHz)d_(ppm): 7.87 (1H, d, J=2 Hz, H-2′), 7.94 (1H, dd, J=2 Hz, 9 Hz, H-6′),7.09 (1H, d, J=9 Hz, H-5′), 7.97 (2H, d, J=9 Hz, H-2″′, 6″′), 7.03 (2H,d. J=9 Hz, H-3″′, 5″′), 6.23 (1H, d, J=2 Hz, H-6), 6.52 (1H, d, J=2 Hz,H-8), 6.68 (1H, s, H=8″), 6.80 (1H, s, H-3 or H3″), 6.83 (1H, s, H-3, or3″), 13.53 (1H, s, HO-5), 13.28 (1H, s, HO-5″), 11.23˜8.63 (4H, br.,4×OH), Anal, Calcd. for C₃₀H₁₈O₁₀.H₂O: C, 64.75; H, 3.62, Found: C, 64.51; H, 3.83.

Improved Procedure for Isolating Robustaflavone (Method No. 1)

[0054] Pigment A (10 g) was dissolved in 50 mL of pyridine. The solutionwas added to 25 g of silica gel and thoroughly mixed. The pyridine wasremoved under reduced pressure using a rotary evaporator and the drymixture ground to a fine particle size. To a 600 mL fritted filterfunnel, incorporating a coarse porosity sinter with a disc of filterpaper placed over the sinter, was added 250 g of silica gel. Theadsorbed Pigment A was then carefully placed and spread on the top ofthe silica gel in the funnel. The solvent system oftoluene/ethanol/formic acid (40:10:2) (2.5 L) was passed through thefunnel to remove the hinokiflavone. The eluent was collected andconcentrated to provide 2.01 g of a yellow solid which was identified ashinokiflavone and a trace of robustaflavone.

[0055] The silica gel in the fritted funnel was allowed to dry outovernight. The top layer containing the adsorbed Pigment A was thenscraped off the remaining silica gel and placed into a fritted filterfuinel of coarse porosity containing a disc of filter paper. The silicagel containing the adsorbed pigment A was then eluted using a mixture oftoluene/ethanol/formic acid (40:10:2) (2.5 L), and then ethanol/pyridine(4:1) (4.5 L). The first eluting solution was concentrated to afford 1.1g of a yellow solid which was identified as a mixture of robustaflavoneand hinokiflavone, the major component being robustaflavone. The secondeluting solution (ethanol/pyridine 4:1) was concentrated to affordrobustaflavone (5.65 g). TLC, NMR, MS, and elemental analysis supportthese findings. NMR (H-NMR, ¹³C-NMR, COSY and HETCOR NMR: see Table 1).

Improved Procedure for Isolating Robustaflavone (Method No. 2)

[0056] Pigment A, a mixture of robustaflavone and hinokiflavone, wasconverted to the hexaacetate and pentacetate, respectively, with aceticanhydride in pyridine. The resulting robustaflavone hexaacetate waspurified by recrystallization from the solvent mixture ofdichloromethane and ethyl acetate to obtain robustaflavone hexaacetatein the yield of 55%. The mother liquor could be further purified bycolumn chromatography to offer additional amounts of robustaflavonehexacetate.

[0057] Robustaflavone hexaacetate was hydrolyzed with 2M aqueous sodiumhydroxide at 60° C. for 30 min. The resulting yellow solution wasfurther stirred at room temperature for additional 2.5 and thenacidified with hydrochloric acid (˜3N) to pH 3 at 0° C. to precipitaterobustaflavone as a yellow solid in a yield of 89.6%.

Robustaflavone Hexaacetate

[0058] To Pigment A (8.84 g) were added 50 mL of pyridine and 50 mLacetic anhydride. The resulting solution was allowed to stand at roomtemperature for 2 days. The reaction mixture was then poured into icewater (800 mL) with stirring. The precipitate was collected byfiltration and rinsed with ice water to remove pyridine and aceticanhydride to afford an ivory solid, 12 g (yield 92.5%), which wasrecrystallized from the solvent mixture of dichloromethane and ethylacetate in the ratio of 9:1 to yield robustaflavone hexaacetate as awhite powder, 6.32 g (55% yield), m.p. 197-198° C., APCI-MS m/z 791.1[M+H]; H-NMR δ_(CDC13) 7.93 (d, J=8.6 Hz, 1H, H-6″′), 7.92 (dd, J-8.7HZ, 2.4 HZ, 2H, H-2′,6′), 7.85 (d,=2.1 HZ, 1H, H-2″′), 7.43 (d, J=2.4Hz, 1H, H-5″′), 7.31 (d, J=2.1 Hz, 1H, H=6″), 7.28 (d, J=2.1 Hz, 1H,H-8″), 6.86 (d, J=2.1 Hz, 1H, H-6″), 6.68 & 6.66 (each s, 1H, H-3,3″),2.44, 2.36, 2.34, 2.21, 2.13 & 2.06 (each, s, 3H, 6x OAc); HPLC Rt 13.31(column: Zorbax 4.6 mm×250 mm, mobile phase: hexane/ethyl acetate 1.2).

Robustaflavone—Hydrolysis of Robustaflavone Hexaacetate

[0059] To robustaflavone hexaacetate (1 g) was added 10 mL of 2M aqueousNaOH and the mixture was heated to 60° C. for 30 min. The resultingyellow solution was further stirred at room temperature for anadditional 2.5 and then acidified with 3 N of hydrochloric acid to pH 3at 0° C. to precipitate robustaflavone. The yellow precipitate wascollected by filtration and washed with ice water to obtainrobustaflavone as yellow solid, 610 mg (yield 89.6%), m.p. 197-198° C.;APCI-MS m/z 539.3 [M+H]⁺; H—NMR and ¹³C—NMR identical to the previousdata; HPLC Rt 42.2 (column: C₁₈, 4.6 mm×15 cm, mobile phase: 20%MeOH/80% of 1% TFA in water to 100% MeOH, gradient).

Characterization of Robustaflavone

[0060] Robustaflavone was recrystallized from pyridine/water, mp.350-352° C. (dec.). The compound gave an orange-red color in the Mg-HCltest and a brown color with alcoholic FeCl₃. The IR spectrum showed abroad hydroxyl absorption at 3250 cm⁻¹ and a conjugated carbonylabsorption at 1650 cm⁻¹. The UV spectrum in MeOH exhibited four maximain the region of 347 (log ε4.38), 300 (4.42), 275 (4.44) and 255 (4.71)nm, and underwent a bathochromic shift on addition of NaOAc or AlC₃. TheUV spectrum in AlCl₃-MEOH was similar to that of in AlCl₃-MEOH uponaddition of HCl, indicating the presence of OH groups at the 5,7 and4′positions, and the absence of an o-dihydroxyl group.

[0061] The NMR spectrum (60 MHz) of robustaflavone exhibited six OHgroups at δ13.53 (s, 1H), 13.28 (s, 1H) and 11.23-8.63 (br, 4H); thefour protons in the 1,4-disubstituted benzene ring appeared at δ7.97 (d,J=9 Hz, 2H) and 7.03 (d, J=9 Hz, 2H); the three protons in the1,3,4-trisubstituted benzene ring appeared at δ7.87 (d, J=9 Hz, 1H),7.94 (dd, J=2 Hz, 9 Hz, 1H) and 7.09 (d, J=9 Hz, 1H); two aromaticprotons appeared as meta-coupled doublets (J=2 Hz) at 6.23 (1H) and 6.52(1H); three isolated protons appeared at d 6.83(s), 6.80(s) and 6.68(s)respectively. The above evidence suggested that the structure of thecompound was composed of two apigenin units joined by an interflavonyllinkage of C3″′-C6, i.e. robustaflavone, an isomer of amentoflavone.This was further supported by examination of its acetate and methylether. Acetylation with pyridine/Ac₂O yielded robustaflavone hexaacetate(3a) as colorless needles, m.p. 199-200° C. Methylation withMe₂SO₄/K₂CO₃ in dry acetone afforded a colorless compound,robustaflavone hexamethylether (3b), m.p. 300-305° C., C₃₆H₃₀O₁₀, M³⁰m/z 622. (The induced change in the chemical shifts (ppm) owing to theaddition of Eu(fod)₃ on compound (3b) is represented by an S-value.³⁵)The S-values of MeO-II-5 and MeO-I-5 were 10.85 ppm (largest) and 2.17ppm respectively, whereas H-I-8 was 0.34 ppm, indicating the presence ofa linkage of CII-3″′-CI-6 as structure (3b) which was characterized ashexa-O-methylrobustaflavone by comparison with an authentic sample (TLC,IR, NMR and MS).³⁵ TABLE I Assignment of ¹³C-¹H HETCOR NMR ¹³C-δ_(ppm)H-δ_(ppm) I-2 164.11^(a) >C═ II-2 163.86^(a) >C═ I-3 102.86 ═CH 6.81(s)II-3 116.10 ═CH 6.84(s) I-4 181.74^(b) >CO II-4 181.83^(b) >CO I-5161.20^(c) ═C—OH 13.02(s) II-5 159.61^(c) ═C—OH 13.23(s) I-6 108.89 ═C<II-6 98.82 ═CH 6.20(d,J=2.0 Hz) I-7 162.06^(d) ═C—OH 10.82-10.00(br)II-7 163.65^(d) ═C—OH 10.82-10.00(br) I-8 93.44 >CH 6.65(s,1H) II-894.05 >CH 6.49(d,J=2.0 Hz) I-9 161.46 ═C—O— II-9 157.5 ═C—O— I-10 103.57═C< II-10 103.72 ═C< I-1′ 121.22 ═C< II-1′ 120.89 ═C< I-2′ 128.55 ═CH7.99(d,J=8.8 Hz) II-2′ 130.87 ═CH 7.79(d,J=2.2 Hz) I-3′ 116.01 ═CH6.96(d,J=8.8 Hz) II-3′ 120.86 ═C< I-4′ 156.35 ═C—OH 10.40(br) II-4′159.07 ═C—OH 10.20(br) I-5′ 116.01 ═CH 6.96(d,J=8.8 Hz) II-5′ 102.86 ═CH7.05(d,J=8.7 Hz) I-6′ 128.55 ═CH 7.99(d,J=8.88 Hz) II-6′ 127.57 ═CH7.93(dd,J=8.7 & 2.2Hz)

[0062] The high resolution CI mass spectrum provided an M+H ion, m/z539.096993, C₃₀H₁₉O₁₀, which requires 539.097821572. The infraredspectrum exhibited a broad hydroxyl absorption at 3250 cm⁻¹ and aconjugated carbonyl absorption at 1650 cm⁻¹. The UV spectrum in MeOHcontained four maxima in the region of 345 (log ε4.49), 300 (4.42), 275(4.44) and 255 (4.71 nm, and underwent a bathochromic shift on additionof NaOAc or AlCl₃. The UV spectrum in AlCl₃-MeOH was similar to thatobtained in AlCl₃-MeOH on addition of HCl, indicating the presence of OHgroups in the 5,7 and 4′ positions, and the absence of an o-dihydroxygroup.²⁶ [l^(NaOAc-MeOH) (log ε) 378 (4.38), 313 (sh 4.41), 277 (4.48),257(4.66) nm; l^(AlC3-MeOH) (log ε) 388 (4.43), 352 (4.50), 300 (4.45),278 (4.45), 254 (4.80 nm).

[0063] The NMR (300 MHz) spectrum of robustaflavone contained six OHgroups at δ13.25 (1H, s), 13.02 (1H, s), 10.83 (1H, s), 10.40 (1H, s),10.4˜10.9 (2H, br.); the four protons in the 1,4-disubstituted benzenering at δ7.98 (2H, d, J=8.88 Hz, H-2″′, 6″′and 6.96 (2H, d, J=8.88 Hz,H-3″′, 5″′); the three protons in the 1,3,4-trisubstituted benzene ringat δ7.93 (1H, dd., J=8.7 Hz and 2.2 Hz, H-6′), 7.79 (1H, d. J=2.2 Hz,H-2′) and 7.05 (1H, d, J=8.7 Hz, H-5′); the five aromatic protons atδ6.84 (1H, s, H-3′), 6.8 (1H, s, H-3″), 6.65 (1H, s, H-8″), 6.49 (1H, d,J=2.0 Hz, H-8) and 6.20 (1H, d, J=2 Hz, H-6).

EXAMPLE 2 General Procedure for Synthesizing O-Acyl Biflavanoids

[0064] Procedure 1: To a solution of biflavanoid in anhydrousdichloromethane containing 20% dry pyridine is added an appropriate acylchloride or anhydride at 0° C. or at room temperature. The mixture isallowed to stand overnight, and the volatiles are evaporated in vacuo.Alternatively, the mixture is poured into water and extracted withchloroform. The organic layer is washed with water and brine, dried overanhydrous sodium sulfate, and concentrated in vacuo. The residue ischromatographed on preparative TLC or a silica gel column to afford theproduct.⁷⁸

[0065] Procedure 2: Preparation of acetate: Biflavanoid is reacted withacetic anhydride in pyridine at room temperature overnight. The reactionmixture is poured into ice water. The precipitate is filtered and washedwith cold 1% hydrochloric acid and then with water to give biflavanoidacetate.³⁷

[0066] Rhusflavanone hexaacetate: Acetylation of rhusflavanone (200 mg)with Ac₂O/Pyridine at room temperature for 20 h gave hexaacetate (110mg) as micro needles, m.p. 130-131° C., EIMS M⁻ m/z 794; IR cm⁻¹ (KBr)1770 (acetoxy CO), 1688 (flavanone CO), 1603, 1560, 1510 and 1490(arom.); H—NMR δ (CDCl₃): 2.02 (3H, s, AcO-7″), 2.10 (3H, s, AcO-7),2.15 (3H, s, AcO-5), 2.28 (3H, s, AcO-4″′), 2.32 (3H, s, AcO-4′), 2.40(3H, s, AcO-5″), 2.85-3.06 (4H, m, H-3.3″), 5.45-5.35 (2H, m. H-2, 2″),6.71 (1H, s, H-6″), 6.91 (1H, s, H-8), 7.14 (2H, d, J=9 Hz, H-3″′, 5″′),7.17 (2H, d, J=9 Hz, H-3′, 5′), 7.44 (2H, d, J=9 Hz, H-2″′, 6″′), 7.55(2H, d, J=9 Hz, H-2′, 6′).

[0067] Succedaneaflavanone hexaacetate: Acetylation ofsuccedaneaflavanone by Procedure No. 2 produced succedaneaflavanonehexaacetate as white needles. m.p. 252-255° C. (from CHCl₃-MeOH), IRcm⁻¹ (KBr): 1770 (OAc), 1688 (flavanone CO), 1613, 1560, 1510 (arom.);H—NMR δ (CDCl₃): 2.10 (6H, s, AcO-7, 7″), 2.17 (6H, s, AcO-5,5″), 2.33(6H, s, AcO-4′, 4″′), 2.83-3.27, 4H, m, H-3, 3″), 5.63 (2H, dd, J=12 Hz,4 Hz, H-2, 2″), 6.97 (2H, s, H-8, 8″), 7.25 (4H, d, J=8 Hz, H-3′, 5′,3″, 5″), 7.58 (4H, d, J=8 Hz, H-2′, 6′, 2″′, 6″′).

[0068] Robustaflavone hexaacetate: A solution of robustaflavone (100 mg,0.186 mmol) in a mixture of pyridine and acetic anhydride (1 mL each)was allowed to stand at room temperature for 72 h. The solution waspoured into ice water, and the resulting white precipitate was collectedon a fritted glass funnel and rinsed with water (123 mg, 84.4%).Following recrystallization from EtOAc/MeOH (1:1, 1.5 mL), 88.5 mg ofoff-white microcrystalline material was obtained. Physical and spectralproperties were identical to those previously reported in theliterature.⁵⁵

EXAMPLE 3 General Procedure for Synthesizing Biflavanoid Ethers

[0069] Preparation of biflavanoid alkyl ethers: To a mixture ofbiflavanoid and Ag₂O (catalytic amount) in DMF is added a correspondingalkyl halide at 10-12° C. After stirring for 2.5-4 h, the reactionmixture is kept in a refrigerator overnight. The catalyst is filtered,and the filtrate is washed with water and brine and then concentrated invacuo. The residue is purified by column chromatography on silica gel toyield the product.⁷⁸

[0070] Preparation of biflavanoid methyl ethers: Biflavanoid isdissolved in anhydrous acetone and potassium carbonate and dimethylsulfate are added. The solution is refluxed for 4 h. The precipitate(potassium carbonate) is filtered and the filtrate is concentrated undervacuum. The residue is dissolved in chloroform and washed with brine,dried with magnesium sulfate and concentrated under vacuum. Theresulting crude product is purified by silica gel column chromatographyor preparative thin layer chromatography and then recrystallized withethyl acetate, ethanol, or chloroform to afford biflavanoid methylethers.³⁷

[0071] Volkensiflavone hexamethyl ether (6)

[0072] Volkensiflavanone (200 mg) was dissolved in 30 mL of anhydrousacetone, and 4 g of potassium carbonate and 3 mL of dimethyl sulfatewere added. The solution was refluxed for 4 h. The precipitate(potassium carbonate) was filtered and the filtrate was concentratedunder vacuum. The reddish brown oily residue was dissolved in 15 mL ofchloroform and the chloroform solution was washed with brine twice andthen water. The chloroform layer was dried with magnesium sulfate andconcentrated under vacuum. The residue was purified by silica gel columnchromatography and eluted with the mixture of toluene and ethyl acetatein the ratio of 1:1. The eluent was concentrated under vacuum and theresidue was recrystallized with methanol/chloroform to obtain 135 mg ofwhite crystals, m.p. 258-260° C., EIMS M⁺ m/z 624; IR cm⁻¹ (KBr): 2900,2950, 2850 (OMe), 1680 (flavanone CO), 1645 (flavone CO), 1600, 1580,1510 and 1490 (arom.) H—NMR d (CDCl₃): 3.93 (3H, s, OMe), 3.87 (3H, s,OMe); 3.83 (6H, s, OMe), 3.77 (3H, s, OMe), 3.67 (3H, s, OMe), 4.90 (1H,d, J=12 Hz, H-3), 5.8 (1H, d, J=12 Hz, H-2), 6.22 (1H, d, J=2 Hz, H-6),6.23 (1H, s, H-6″′), 6.32 (1H, d, J=2 Hz, H-8), 6.50 (1H, s, H-3″), 6.63(1H, s, J=9 Hz, H-3′, 5′), 6.87 (2H, d, J=9 Hz, H-3″′, 5″′), 7.13 (2H,d, J=9 Hz, H-2′, 6′), 7.70 (2H, d, J=9 Hz, H-2″′, 6″).

[0073] GB-1a hexamethyl ether (14)

[0074] GB-1a (200 mg) was methylated by the method described above. Theresulting crude methyl ether was purified by preparative thin layerchromatography using ethyl acetate as developing solvent. The band at Rf0.35 was scraped off and extracted with ethyl acetate. The ethyl acetateextract was concentrated under vacuum and the residue was recrystallizedfrom the solvent mixture of acetone and hexane (1:1) to afford a whitesolid, 118 mg, m.p. 132-134° C., EIMS M⁺ m/z 626, IR cm⁻¹ (KBr), 2990,2930, 2900, 2830 (OMe); 1675 (flavanone CO), 1600, 1570 and 1515 cm⁻¹(arom.); H—NMR δ(CDCl₃): 2.72 (2H, m, H-3″), 3.90 (6H, s, 233 OMe), 3.83(6H, s, 2×OMe), 3.90 (6H, s, 2×OMe), 4.70 (1H, d, J=12 Hz, 3-H), 5.28(1H, m, H-2″), 5.73 (1H, d, J=12 Hz, H-2), 6.08 (1H, d J=2 Hz, H-6),6.15 (1H, s, H-6″), 6.17 (1H, d, J=2 Hz, H-8), 6.82 (2H, d, J=8 Hz,H-3′, 5′), 6.90 (2H, d, J=8 Hz, H-3″′, 5″′), 7.28 (2H, d, J=8 Hz, H-2′,6′), 7.32 (2H, d, J=8 Hz, H-2″′, 6″′).

[0075] Robustaflavone hexamethyl ether. A solution of robustaflavone(200 mg, 0.272 mnol) in acetone (20 niL) and dimethylsulfate (2 mL)containing 3.8 g potassium carbonate was heated at reflux for 72 h withmagnetic stirring. Filtration followed by evaporation of the filtrateafforded a black, oily semi-solid. Purification via columnchromatography (silica gel, CHCl₃/MeOH, 98:2) provided the desiredproduct as an off-white solid (140 mg, 60.6%). Physical and spectralproperties were identical to those previously reported in theliterature.⁵⁵

EXAMPLE 4 General Procedure for Preparation of Biflavanoid Sulfates

[0076] The dicyclohexylcarbodiimide (DDC)-mediated esterification offlavones and flavonols with tetrabutylammonium hydrogen sulfate (TBSHS)resulted in the formation of mono-, di-, and trisulfated products bycontrolling the reaction temperature and amount of reagents. Sulfationoccurred mainly at positions 7,4′ and 3 of the flavonoid skeleton andfollowed the order 7>4′>3.⁸⁰ Biflavanoid partial sulfate esters areprepared by treating the biflavanoid with TBAHS (tetrabutylammoniumhydrogen sulfate) and DDC (dicyclohexylcarbodiimide) in pyridine usingcontrolled amounts of reagents and temperature. The reaction product,sulfate ester TBA-salt, is separated from minor by-products by gelfiltration. The sulfate ester TBA-salt is converted to the potassiumsalt by treatment with saturated methanolic potassium carbonate. Theresulting potassium salt is purified by repeated chromatography onSephadex G-10 column using a 0-50% gradient of aqueous methanol.^(λ)

Robustaflavone Tetrasulfate K-salt

[0077] A solution of robustaflavone (46.1 mg, 0.086 mM, 1.0 equivalent)in pyridine (5 mL) was treated with 1,3-dicyclohexylcarbodiimide (DCC)(500 mg, 2.423 mM, 28.17 equivalent) and tetrabutylammonium hydrogensulfate (TBAHS) (97.5 mg, 0.287 mM, 3.34 equivalent) at 4° C. (inrefrigerator) for 86 hours. The reaction solution was diluted with MEOHand the dicyclohexylurea precipitate was removed by filtration. Thesupernatant was chromatographed on Sephadex LH-20 (3 g, in MEOH) andeluted with MEOH and a MeOH-acetone (1:1) mixture. The yellow fractionscontaining robustaflavone tetrasulfate were concentrated to 5 mL andthen treated with 15 mL of saturated K₂CO₃ in MEOH. The precipitate ofrobustaflavone tetrasulfate K-salt was collected by filtration andwashed with MEOH (3ml×9) and water 3 mL×5), successively. The MEOH andwater washes were collected separately. The water solution waslyophilized to obtain 72 mg robustaflavone-7,4′,7″,4″′-tetrasulfateK-salt as a yellow powder, ¹H—NMR (DMSO, 300 MHz) δ6.56 (1H, bs, H-6),7.19 (1H, bs, H-8), 6.78 (1H, s, H-3), 7.75 (1H, dd, J=9.0, 2.0 Hz,H-6′), 7.87 (1H, d, J=9.0 Hz, H-5′), 8.31 (1H, d, J=2.0 Hz, H-2′), 6.85(1H, s, H-8), 6.75 (1H, s, H-3″), 7.33 (2H, d, J=9.0 Hz, H-3″′, 5″′),7.94 (2H, d, J=9.0 Hz, H-2″′, 6″′).

EXAMPLE 5 General Procedure for Preparation of Biflavanoid Acid Salt

[0078] The dried mixture of biflavanoid, appropriate acid anhydride, andappropriate catalyst, such as 4-dimethylaminopyridine are dissolved indry pyridine. The solution is worked-up by standard methods to yieldbiflavanoid acid adduct. The biflavanoid acid can be converted to thepotassium salt by treatment with saturated methanolic potassiumcarbonate.⁷⁹

Robustaflavone Tetrasodium Salt

[0079] Robustaflavone (53.8 mg, 0.10 mmol) was dissolved in 0.400 mL of1.0 M NaOH, and the resulting dark yellow solution was freeze dried.Following drying, 62 mg (99% of theoretical yield) of a brick-orangeglass was obtained. The product is believed a mixture of salt formswhich may include the tetrasodium salt form. Robustaflavone tetrasodiumsalt was assayed for activity and was approximately 10-fold less activethan robustaflavone in the vitro assay against HBV (see Example 6). Itwas also moderately active against adenovirus type 1:EC₅₀ =32 uM;IC₅₀>160 uM.

EXAMPLE 6 Antiviral HBV Activity of Biflavanoids

[0080] In this example, robustaflavone and related biflavanoids werescreened for hepatitis B (HBV) antiviral and cytotoxicity activity.

[0081] Antiviral HBV Assay. The inhibition of HBV replication incultures of 2.2.15 cells was assayed using chronically HBV-producinghuman liver cells which were seeded into 24-well tissue culture platesand grown to confluence. Test compounds were added daily for a ninecontinuous day period; the culture medium was collected and stored foranalysis of extracellular (virion) HBV DNA after 0, 3, 6, and 9 days oftreatment. The treated cells were lysed 24 hours following day 9 oftreatment for the analysis of intracellular HBV genomic forms. Theoverall levels of HBV DNA (both extracellular and intracellular DNA) andthe relative rate of HBV replication (intracellular DNA) were analyzedquantitatively. The analysis was performed using blot hybridizationtechniques and [³²P]-labeled HBV-specific probes. The HBV DNA levelswere measured by comparison to known amounts of HBV DNA standardsapplied to every nitrocellulose membrane (gel or slot blot). An AMBISbeta scanner, which measures the radioactive decay of the hybridizedprobes directly from the nitrocellulose membranes, was used for thequantitative analysis. Standard curves, generated by multiple analyses,were used to correlate CPM measurements made by the beta scanner withrelative levels of target HBV DNA. The levels of HBV virion DNA releasedinto the culture medium were analyzed by a slot blot hybridizationprocedure. HBV DNA levels were then compared to those at day 0 todetermine the effect of the test compound. A known positive drug wasevaluated in parallel with test compounds in each test. This drug was2′,3′-dideoxycytosine (2′,3′-ddC) or lamivdine (3TC). The data wereexpressed as 50% effective (virus-inhibitory) concentrations (EC₅₀). The90% effective concentration (EC₉₀), which is that test drugconcentration that inhibits virus yield by 1 log₁₀, was determined fromthese data. Each test compound's antiviral activity was expressed as aselectivity index (SI), which is the CC₅₀ or CC₉₀, the concentration ofcompound which killed 50% or 90% of the treated cells, divided by theEC₅₀. Generally an SI of 10 or greater is indicative of positiveantiviral activity, although other factors, such as a low SI for thepositive control, are also taken into consideration.

[0082] HBV Cytotoxicity Assays. The toxicity of the test compounds incultures of 2.2.15 cells, grown to confluence in 96-well flat-bottomedtissue culture plates and treated with compounds as described above,were assayed at four concentrations each in triplicate cultures, in 3 to10-fold steps. Untreated control cultures were maintained on each plate.On each plate, wells containing no cells were used to correct for lightscattering. The toxicity was determined by the inhibition of the uptakeof neutral red dye, determined by absorbance at 510 nm relative tountreated cells, 24 hours following day 9 of treatment.

[0083] Analysis of HBV Nucleic Acids and Proteins. HBV viron DNA inculture medium, and intracellular HBV RI and HBV RNA levels weredetermined by quantitative blot hydridization analyses (dot, Southern,and Northern blot, respectively).^(81,82) Nucleic acids were prepared bypreviously described procedures. Integrated HBV DNA, which remains at astable level per cell during the treatment periods was used toquantitate the amount of cellular DNA transferred in each Southern gellane.^(81,82) For the HBV RNA analyses, the levels of b-actin RNA wereused to quantitate the amount of cellular RNA transferred in eachNorthern gel lane. Previous examinations of b-actin-specific RNA inconfluent cultures of 2.2.15 cells demonstrated a steady state level ofapproximately 1.0 pg b-actin RNA/mg unfractionated cellular RNA.⁸¹ EC₉₀values (10-fold depression of HBV DNA levels relative to untreatedcontrol cultures) were determined by linear regression. ⁸² EC₉₀ valueswere used for comparison since, in this culture system, DNA levelswithin 3-fold of control values are not generally statisticallysignificant.⁸³

[0084] Values of HBV proteins were determined by semi-quantitative EIAperformed as previously described.⁸³ For the EIA analyses, test sampleswere diluted (2- to 10-fold) so that the assay values produced werewithin the linear dynamic range of the EIA assays. Standard curves usingserial dilutions of positive assay controls were included in each set ofEIA analyses. HBV surface antigen (HBcAg), preSI protein, and HBcantigen (HBcAg) are released as extracellular products and weretherefore analyzed in culture medium obtained 24 h following the lasttreatment dose of oligonucleotides or 2′, 3′-ddc. HBV core antigen(HBcAg) is an intracellular viral protein and was assayed in cellextracts produced by Triton-X-100 lysis.⁸³

[0085] Cultures for HBV RNA were maintained on 6-well plates, culturesfor HBV virion DNA analyses were maintained on either 96- or 24-wellplates, and cultures for all other HBV parameters were maintained on24-well plates.

[0086] The concentrations of antiviral agents used in these studiesapproximates the EC₅₀ values of the individual agents againstintracellular HBV DNA replication intermediates (HBV RI). Cultures weretreated with the indicated agents for 9 days using standard procedures.Values reported are the levels of the indicated HBV markers at the endof the treatment period (“DAY 9”) expressed as a percentage (±standarddeviation (S.D.) of the average levels in the control cultures at thebeginning of the treatment period (“DAY 0”). The method of expressionpermits an analysis of the variation of the HBV markers in the untreated(control) cultures over the course of the treatment period. HBV nucleicacid levels were measured by standard blot hydridization (dot, Southern,or Northern). HBV protein levels were measured by standardsemi-quantitative ELA methods. Cultures for HBV RNA were maintained in6-well culture plates. The levels of each of two major classes of HBVRNA transcripts are listed separately. Cultures for all other HBVmarkers were maintained in 24-well culture plates. For each treatment, atotal of 4 separate cultures were used for the analysis of each HBVmarker at both DAY 0 and DAY 9.

[0087] Results. Tables 2 and 3 present evidence that robustaflavone isan extremely effective anti-HBV agent against the hepatitis B virus incomparison to the control drug, 2′,3′-ddC. It was observed from theresults that robustaflavone exhibited an impressive in vitro activityagainst extracellular (virion) HBV DNA, with an effective averageconcentration (EC₅₀) of 0.25 uM and an average selectivity index(CC₅₀/EC₉₀) of 153; compared to an effective average EC₅₀ of 1.4 uM andaverage SI of 31 for 2′,3′-ddC. Furthermore, measurement of the relativerate of HBV replication intermediates (RI) (intracellular DNA) againindicates the effectiveness of robustaflavone over the control drug,2′,3′-ddC. Robustaflavone exhibits an effective EC₅₀ of 0.6 uM and SI of80; compared to an EC₅₀ of 2.4 uM and SI of 24 for 2′,3′-ddC.Volkensiflavone hexamethyl ether (6), rhusflavanone acetate (10) andsuccendaneaflavanone hexaacetate (12) exhibited moderate anti-HBVactivity while amentoflavone (1), agathistflavone, hinokiflavone (4),volkensiflavone (5), rhusflavanone (9) and succendaneaflavanonepossessed little or no anti-HBV activity.

[0088] In summary, measurement of the overall levels of HBV DNA (bothextracellular and intracellular DNA) and the relative rate of HBVreplication intermediate (RI) (intracellular DNA) clearly demonstratesthe effectiveness of robustaflavone against HBV. TABLE 2 Anti- HBVactivity of biflavanoids, biflavanones, and related semi-syntheticderivatives Hepatitis B Virus (HBV) HBV Virion EC₅₀ ¹ EC₉₀ ² SI³ SampleμM μM (CC₅₀/EC₉₀) 2′,3′-ddC* 1.8 9.4 28 Lamivudine (3TC) 0.038 0.1611200 Penciclovir 0.19 0.92 471 Robustaflavone 0.25 2.2(5.6) 153(60)(0.60) Robustaflavone 2.9 28 >36 hexamethyl ether Robustaflavone 0.732.8(11) >360(163) Hexaacetate (3.9) Amentoflavone (1) >100 >100 NDAgathisflavone (2) >100 >100 ND Robustaflavone (3) 0.25 2.4 153Hinokiflavone (4) >100 >100 ND Volkensiflavone (5) >100 >100 NDVolkensiflavone 11 108 13 Hexamethylether (6) Rhusflavanone(9) >100 >100 ND Rhusflavanone 7.1 6.2 2.8 hexaacetate (10)Succedaneaflavanone (11) >100 >100 ND Succedaneaflavanone 3.5 128 1.9hexaacetate (12) Robustaflavone sodium 3.1 12 88 salt Robustaflavone 0.43.6 110 tetrasulfate (17)

[0089] TABLE 3 Effect of Antiviral Agents on HBV Proteins and NucleicAcids in 2.2.15 Cells Relative Levels of HBV Proteins^(a) and NucleicAcids (Day 9, % of Day 0 Control ± SD) Viral DNA Viral mRNA Extra-Intra- 3.6 kb and 3.6 kb and cellular cellular 2.1 kb 2.1 kb ViralAntigens Treatment DNA DNA mRNA mRNA HBsAg HBeAg HbcAG Untreated 127 ±8  103 ± 11  90 ± 12 101 ± 10 117 ± 11 108 ± 5   86 ± 10 cells 2′,3′-ddC1 + 1 6 + 1 94 + 7  87 + 9  90 − 12  88 + 10 91 + 9 Robusta- 1 ± 1 5 ± 193 ± 10 106 ± 11 97 ± 6 86 ± 6 138 ± 8  flavone @ 10 μM

EXAMPLE 7 Antivirial HBV Activity of Combinations of Robustaflavone, 3TCand PCV

[0090] Lamivudine and penciclovir were purchased from MoravekBiochemical (La Brea, Calif., U.S.A.). Robustaflavone was isolated fromthe seeds of Rhus succedanea, as described above.

[0091] Assay of anti-HBV activity and toxicity in 2.2.15 cells.Determination of therapeutic concentrations (EC₅₀ and EC₉₀) and toxicityconcentrations (IC₅₀) were determined as previously described (90).Stock solutions of text drugs were prepared in DMSO. Culture medium waschanged daily and analyzed for extracellular HBV DNA after nine days ofcontinuous drug exposure. Extracellular HBV DNA was extracted from theculture media and analyzed by a slot blot hydridization technique, usinga ³²P-labeled Eco RI HBV DNA fragment, as previously described (91), andquantitated via comparison to HBV standards on a nitrocellulose filter,using an AMBIS beta scanner. Toxicity was determined by measuringinhibition of neutral red dye uptake. Effective concentration (EC₅₀ andEC₉₀) and toxicity (IC₅₀) values were calculated via comparison withdrug-free controls. Effective concentration values for individual agentsare the mean of six cultures per concentration point. Toxicity valuesare the mean of three cultures per concentration point.

[0092] Combination Studies of Robustaflavone, 3TC and PCV in 2.2.15cells. Cultures were treated with combination of agents as previouslydescribed (92). Briefly, antiviral agents were mixed at approximatelyequipotent molar concentrations based on the EC₅₀ values. Serialdilutions of these mixtures were then used to treat cultures asdescribed above along with the appropriate monotherapies. For thesestudies, eight cultures were used for each of six 3-fold dilutions.

[0093] Determination of Mechanism of Action. Measurements ofintracellular and extracellular HBV DNA, 3.6 kb and 2.1 kb mRNAfragments, and HBsAg, HBeAg and HBcAg protein markers were conducted aspreviously described (93).

[0094] Results and Discussion. Robustaflavone exhibited activity againstHBV replication in a chronically infected (90) human hepatoblastoma cellline (2.2.15), inhibiting the replication of HBV by 50% relative todrug-free controls at a concentration of 0.25 μM (EC₅₀, mean of twotrials), with an in vitro therapeutic index (TI, IC50/EC90) of 153(Table 2). None of the other compounds tested from the series ofbiflavanoids showed significant activity against HBV with an acceptableselectivity index (Table 2); thus, robustaflavone was the onlyderivative selected for further evaluation. In a comparison with severalnucleoside antiviral agents, the anti-HBV activity of robustaflavone wassuperior to ddC (EC₅₀=1.4 μM, TI=30) and similar to penciclovir(EC₅₀=0.19 μM, TI=471). Lamivudine was clearly the most active of theagents evaluated (EC₅₀=0.038 μM, TI=11200).

[0095] Combinations of antiviral agents are now accepted to be superiorto monotherapy in certain instances, particularly in treatment of HIVinfection, because of the ability of drug combinations to overcomeresistant mutants which accumulate following exposure to a single agent(89). Clinical isolates of 3TC-resistant mutants of HBV have recentlybeen reported following monotherapy with that agent (87,88).

[0096] Robustaflavone was evaluated in combination with two leadinganti-HBV candidates, 3TC and penciclovir (the bioactive form offamciclovir), in an effort to determine if combinations ofrobustaflavone with either of these drugs could potentially offersynergistic benefits if used as part of an anti-HBV regimen. As shown inTable 4, combinations of robustaflavone (RF) with 3TC at differingratios showed varying degrees of syngergism and antagonism. It isinteresting to note that the most effective ratio of RF to 3TC was 10:1,which exhibited an EC₅₀ of 0.054 μM with respect to RF (0.0054 μM withrespect to 3TC). The ratio of 10:1 correlates well with the EC₅₀ ratiofor RF and 3TC (RF/3TC=6.6). The concentration of 3TC in the 10:1combination (0.038 μM) necessary to achieve 90% reduction of HBVreplication was lower than that required by 3TC alone (0.16 μM),illustrating the potential benefits of the combination. Ratios ofRF:30:1 and 3:1 were less effective than the 10:1 conbination.Combostat® analysis⁹⁶ of the RF/3Tccombination data indicated that, at amolar ratio of 10:1, the combination exhibited syngergism at alldilutions. The RC:3TC ratio of 30:1 exhibited antagonism at lower drugconcentrations, and additive or mildly syngergistic effects at higherconcentrations. At a RF:3TC ratio of 3:1, the combination exhibitedantagonism at all drug concentrations.

[0097] The combinations of RF with penciclovir (PCV) also showed a clearsyngergistic effect. Because RF and PCV have similar effectiveconcentrations (EC₅₀ values of 0.25 and 0.19 μM, respectively) andcellular toxicity concentrations (IC₅₀ values of 337 and 433 μM,respectively), the synergistic effects of the agents together was muchmore obvious. At a RF:PCV ratio of 10:1, the selectiveity index wasinferior to either drug alone. At a RF:PCV ratio of 3:1, the selectivityindex improved to 570, higher than either drug alone, while a RF:PCVratio of 1:1 exhibited an even higher selectivity index of 684. At the1:1 combination ratio, the concentration of PCV necessary to achieve 90%reduction of HBV replication was 0.51 μM, nearly two-fold less than theconcentration required of PCV alone (0.92 μM). Combostat® analysis ofthe combination data indicated that a RF:PCV ratio of 1:1 exhibitedsynergism at all concentrations. At a RF:PCV ratio of 3:1, thecombination still showed a mild degree of syngergism at allconcentrations except for the very lowest, while a 10:1 ratio exhibitedantagonism at most concentrations. Again, the ratio exhibiting thegreatest syngergistic effect was that which mirrored the EC₅₀ ratio forthe two agents (RF/PCV=1.3).

[0098] To determine the likely mechanism of action for robustaflavone,levels of extracellular and intracellular HBV DNA, mRNA and proteinantigen markers were measured in 2.2.15 cells on day nine followingcontinuous treatment (93). As presented in Table 3, the levels ofextracellular and intracellular HBV DNA were dramatically decreasedrelative to drug-free controls for both robustaflavone and ddC, whichwas used as a positive control. Neither the levels of viral mRNA (3.6and 2.1 kb) nor the three major viral antigens (HBsAg, HBeAg and HBcAg)were significantly affected by exposure of the cells to robustaflavone.These results strongly suggest that robustaflavone acts via inhibitionof the viral DNA polymerase, supported by the fact that results forrobustaflavone were essentially identical to those for ddC, anestablished inhibitor of viral polymerases, used as a positive control.

[0099] In conclusion, robustaflavone represents a novel non-nucleosidenatural product possessing impressive activity against hepatitis B virusreplication, determined in a chronically replicating transfected humancell line. The paucity of agents available for the treatment of HBVinfection underscore the need for development of new lead compounds,especially non-nucleoside structures, which would complement the drugscurrently in development. TABLE 4 Antiviral and cytotoxicity effects ofrobustaflavone (RF), lamivudine (3TC), penciclovir (PCV) and drugcombinations in 2.2.15 cells^(a) Drug EC₅₀ EC₉₀ IC50 TI 3TC or (orcombination) (μM)^(b) (μM)^(b) (μM) (IC₅₀/EC₉₀) PCV (μM)^(c) 3TC 0.0380.16 1790 11200 — PCV 0.19 0.92 433 471 — RF 0.25 2.2 337 153 — RF + 3TC(30:1) 0.33 1.7 325 191 0.057 RP + 3TC (10:1) 0.054 0.38 340 894 0.038RF + 3TC (3:1) 0.16 0.73 363 497 0.24 RF + PCV (10:1) 0.99 2.9 334 1150.29 RF + PCV (3:1) 0.22 0.63 360 570 0.21 RF + PCV (1:1) 0.11 0.51 349684 0.51

EXAMPLE 8 Anti-Respiratory Viral Activity of Biflavanoids

[0100] In this example, robustaflavone and related biflavanoids werescreened for respiratory (influenza A and B, RSV, parainfluenza 3,adenovirus 5, and measles) antiviral and cytotoxic activities.

[0101] Anti-Respiratory Viral Assay. The viruses used in the primaryscreen for antiviral activity against respiratory viruses consisted of:(1) Influenza A and B—Virus strains: A/Texas/36/91 (H1N1) (Source:Center for Disease Control (CDC), A/Beijing/2/92 (H3N2) (Source: CDC),B/Panama/45/90 (Source: CDC), A/NWS/33 (H1N1) (Source: American TypeCulture Collection [ATCC]). (All but A/NWS/33 are tested in the presenceof trypsin.); cell lines: Madin Darby canine kidney (MDCK) cells; (2)Respiratory syncytial virus—Virus strain: Utah 89 (Source: Utah StateDiagnostic Laboratory, cell line: African green monkey kidney (MA-104)cells; (3) Parainfluenza type 3 virus—Virus stain: C243 (Source: ATCC);cell line: African green monkey kidney (MA-104) cells; (4) Measlesvirus—Virus strain: CC (Source: Pennsylvania State University; cellline: African green monkey kidney (BSC-1) cells; and (5) Adenovirus type5—Virus strain: Adenoid 75 (Source: ATCC); cell line: Human lungcarcinoma (A549) cells.

[0102] Test compounds were assayed for continual activity andcytotoxicity. Three methods were used for assay of antiviral activity:(1) inhibition of the viral cytopathic effect (CPE); (2) increase in theneutral red (NR) dye uptake; and (3) decrease in the virus yield.Methods for ascertaining cytotoxicity were visual observation, neutralred uptake, and viable cell count.³²

[0103] Inhibition of the Viral Cytopathic Effect (CPE). The test for CPEwas run in 96-well flat-bottomed microplates and was used for theinitial antiviral evaluation of all new test compounds. In this CPEinhibition test, seven one-half log₁₀ dilutions of each test compoundwere added to 4 cups containing the cell monolayer; within 5 min, thevirus was then added and the plate sealed, incubated at 37° C. and CPEread microscopically when untreated infected controls develop a 3 to 4+CPE (approximately 72 h). A known positive drug was evaluated inparallel with test drugs in each test. This drug was ribavirin forinfluenza, measles, respiratory syncytial, and parainfluenza viruses,and (S)-1-(3-hydroxy-2-phosophonylmethoxypropyl)adenine (HPMPA) foradenovirus. The data were expressed as 50% effective (virus-inhibitory)concentrations (EC₅₀).

[0104] Increase in the Neutral Red (NR) Dye Uptake. The test forincrease in the NR dye uptake was run to validate the CPE inhibitionseen in the initial test, and utilizes the same 96-well microplatesafter the CPE has been read. Neutral red dye was added to the medium;cells not damaged by virus take up a greater amount of dye, which wasread on a computerized microplate autoreader. An EC₅₀ value wasdetermined from this dye uptake.

[0105] Decrease in virus yield. Compounds considered active by CPEinhibition and NR uptake were retested using both CPE inhibition, and,using the same plate, the effect on reduction of virus yield wasdetermined by assaying frozen and thawed eluates from each cup for virustiter by serial dilution onto monolayers of susceptible cells.Development of CPE in these cells was an indication of presence ofinfectious virus. As in the initial tests, a known active drug(ribavirin) was run in parallel as a positive control. The 90% effectiveconcentration (EC₉₀), which was that test drug concentration thatinhibits virus yield by 1 log₁₀, was determined from these data.

[0106] Cytotoxicity Assays. These assays consist of visual observation,neutral red dye uptake, and viable cell count.

[0107] Visual Observation—In the CPE inhibition tests, two wells ofuninfected cells treated with each concentration of test compound wererun in parallel with the infected, treated wells. At the same time CPEwas determined microscopically, the toxicity control cells were examinedmicroscopically for any changes in cell appearance compared to normalcontrol cells run in the same plate. These changes were given adesignation conforming to the degree of cytotoxicity seen (e.g.,enlargement, granularity, cells with ragged edges, a cloudy appearance,rounding, detachment from the surface of the well, or other changes.These changes were given a designation of T (100% toxic), Pvh (partiallytoxic-very heavy 80%), Ph (partially toxic-heavy 60%), P (partiallytoxic-40%), Psi partially toxic-slight-20%), or 0 (no toxicity −0%),conforming to the degree of cytotoxicity seen. A 50% cell inhibitory(cytotoxic) concentration (IC₅₀) was determined by regression analysisof the data.

[0108] Neutral Red Dye Uptake—In the neutral red dye uptake phase of theantiviral test described above, the two toxicity control wells alsoreceive neutral red dye and the degree of color intensity was determinedspectrophotometrically. A neutral red IC₅₀ was subsequently deternmined.

[0109] Viable Cell Count—Compounds considered to have significantantiviral activity in the initial CPE and NR tests were retested fortheir effects on cell growth. In this test, 12-well tissue cultureplates were seeded with cells (sufficient to be approximately 20%confluent in the well) and exposed to varying concentrations of the testdrug while the cells were dividing rapidly. The plates were thenincubated in a CO₂ incubator at 37° C. for 72 h, at which time themedia-drug solution was removed and the cells washed. Trypsin was addedto remove the cells, which were then counted using a Coulter cellcounter. An IC₅₀ was then determined using the average of three separatecounts at each drug dilution.

[0110] Each test compound's antiviral activity was expressed as aselectivity index (SI), which was the IC₅₀ or IC₉₀ divided by EC₅₀.Generally an SI of 10 or greater was indicative of positive antiviralactivity, although other factors, such as a low SI for the positivecontrol, were also taken into consideration.

Anti-Influenza A and Anti-Influenza B Activity

[0111] Compounds 1-6 and 9-12 have been screened for inhibitory activityagainst influenza A (strains H1N1 and H3N2) and influenza B viruses. Forthese compounds both cytopathic effect inhibition (CPE) and neutral reduptake test methods were investigated. The results are displayed onTables 5-7. For the results shown in Tables 5-7 the selective index (SI)is calculated as IC₅₀ (50% cell inhibitor (cytotoxic) concentration)over the EC₅₀ (50% effective concentration).

[0112] Influenza A. Tables 5 and 6 provide data that robustaflavone (3)had significant antiviral activity towards two influenza A strains, whencompared to the control drug, ribavirin. The effective concentrations(EC₅₀) of robustaflavone (3) were 1.9 μg/mL for both influenza AH1N1(Table 5) and H3N2 (Table 6) strains, as compared to 1.9 and 4.1ug/mL for the control drug, ribavirin. The IC₅₀ values forrobustaflavone were 18 and 32 ug/mL, respectively for H1N1 and H3N2 inthe CPE assay. However, the selectivity indexes (SI) for ribavirin were296 and 137 against influenza A strains H1N1 and H3N2, respectively, ascompared to 9.5 and 17 for robustaflavone (3). The effective neutral redconcentrations (EC₅₀) of robustaflavone (3) were 2.0 and 1.8 ug/mL forinfluenza A strains H1N1 and H3N2, respectively and the IC₅₀ values were˜32 and ˜100 ug/miL. This compared favorably with ribavirin, which hadeffective neutral red concentrations of 1.4 and 5.7 ug/mL, respectivelyfor these strains. The SI's for neutral red uptake for ribavirin were132 and 70, respectively, toward influenza A strains H1N1 and H3N2,whereas those for robustaflavone (3) were 16 and 56.

[0113] Amentoflavone (1) also demonstrated significant antiviralactivity against both strains of influenza A. The EC₅₀ values ofamentoflavone (1) were 3.1 and 4.3 μg/mL, respectively in CPE inhibitiontests. The IC₅₀ values were 22 and >100 μg/mL, therefore it had SIvalues of 7.1 and >23 for influenza A strains H1N1 and H3N2. The otherbiflavanoids assayed were either inactive or toxic, except foragathisflavone which produced an SI value of >18 for the neutral redassay, but only 1 for the CPE assay. The acetylation of rhusflavanone(9), to rhusflavanone hexaacetate (10), slightly increased both theactivity and toxicity against both influenza A strains in both assays.The acetylation of succedaneaflavanone (11) did not change the activityor toxicity considerably, and methylation of volkensiflavone (5) tovolkensiflavone hexamethyl ether (6) resulted in a decrease in both theactivity and the toxicity, in both the CPE inhibition and the neutralred assays. As shown in Tables 5 and 6, the modifications to these threecompounds did result in changes in activity and toxicity, but noneproduced significant changes in the SI value. TABLE 5 Influenza A (H1N1)Virus: Texas/36/91 CPE Inhibition Neutral Red EC₅₀*¹ IC₅₀*² EC₅₀*¹IC₅₀*² Sample ug/mL ug/mL SI*³ ug/mL ug/mL SI*³ Ribavirin* 1.9 562 2961.4 185 132 Amentoflavone (1) 3.1 22 7.1 5.3 >100 19 Agathisflavone (2)6.6 6.5 1.0 5.6 >100 18 Robustaflavone (3) 1.9 18 9.5 2.0 32 16Hinokiflavone (4) >1.0 1.4 <1.4 1.8 2.0 1.1 Volkensiflavone (5) >32 13 015 14 1.0 Volkensiflavone ˜100 <24 0 ˜100 ˜100 0 hexamethyl ether (6)Rhusflavanone (9) >10 8.2 0 24 26 1.1 Rhusflavanone >10 7.2 0 5.6 5.71.0 hexaacetate (10) Succedanea- >3.2 4.9 <1.5 5.2 5.0 1.0 flavanone(11) Succedanea- 5.6 8.2 1.5 7.4 7.4 1.0 flavanone hexaacetate (12)

[0114] TABLE 6 Influenza A (H3N2) Virus: Beijing/32/92 CPE InhibitionNeutral Red EC₅₀*¹ IC₅₀*² EC₅₀*¹ IC₅₀*² Sample ug/mL ug/mL SI*³ ug/mLug/mL SI*³ Ribavirin* 4.1 562 137 5.7 397 70 Amentoflavone (1)4.3 >100 >23 6.5 >100 >15 Agathisflavone (2) 24 18 0.8 13 19 1.5Robustaflavone (3) 1.9 ˜32 17 1.8 ˜100 56 Hinokiflavone (4) >3.2 1.3 01.9 2.2 1.2 Volkensiflavone (5) 56 42 0.8 38 37 1.0 Volkensiflavone ˜100˜100 0 ˜100 ˜100 0 hexamethyl ether (6) Rhusflavanone (9) >32 24 0 31 311.0 Rhusflavanone >10 5.6 0 5.4 5.3 1.0 hexaacetate (10) Succedanea- >1012 <1.2 12 12 1.0 flavone (11) Succedanea- 8.8 12 1.4 5.6 5.6 1.0flavone (12)

[0115] Influenza B. Table 7 indicates that robustaflavone hadsignificant antiviral activity towards influenza B, when compared to thecontrol drug, ribavirin. The effective concentration (EC₅₀) ofrobustaflavone was an impressive 0.23 ug/mL, compared to 1.5 forribavirin. The selectivity index (SI) for ribavirin was >667 againstinfluenza B; as compared to <435 for robustaflavone. The effectiveneutral red concentration (EC₅₀) of robustaflavone was 0.22 ug/mL,compared to the control drug, ribavirin, 0.48 ug/mL. The SI for neutralred uptake for ribavirin was 208, compared to 454 for robustaflavone.TABLE 7 Influenza B Virus: Panama/45/90 CPE Inhibition Neutral RedEC₅₀*¹ IC₅₀*² EC₅₀*¹ IC₅₀*² Sample ug/mL ug/mL SI*³ ug/mL ug/mL SI*³Ribavirin* 1.5 >1000 >667 0.48 100 208 Amentoflavone 0.56 100 178 — — —(1) Agathisflavone 3.2 18 5.6 — — — (2) Robustaflavone 0.23 ˜100 ˜4350.22 ˜100 454 (3) Hinokiflavone (4) >1.0 1.2 <1.2 1.9 2.0 1.0Volkensiflavone 1.1 38 34 4.5 20 4.4 (5) Volkensiflavone 2.6 ˜100 ˜38<20 ˜100 5.0 hexamethyl ether (6) Rhusflavanone >4.1 38 9.3 — — — (9)Rhusflavanone >10 4.2 0 — — — hexaacetate (10) Succedanea- 0.97 15 152.2 7.0 3.2 flavanone (11) Succedanea- 5.4 12 2.2 5.9 5.9 1.0 flavanonehexaacetate (12)

[0116] Amentoflavone (1) (I-3′-II-8 biapigenin), volkensiflavone (5)(naringenin I-3-II-8 apigenin), volkensiflavone hexamethyl ether andsuccedaneaflavanone (11) (I-6-II-6 binaringenin) also exhibitedfavorable antiviral activity against influenza B, having SI values of178, 34, 38, and 15, respectively in the CPE assay. Agathisflavone(2)(I-6-11-8 biapigenin) and rhusflavanone (9) (I-6-II-8 binaringenin)demonstrated activity against influenza B virus, with SI values of 5.6and 9.3, for the CPE assay. However in neutral red uptake tests, thesebiflavanoids showed no significant activity. None of the otherbiflavinoids assayed contributed significant activity. Methylation ofvolkensiflavone (5), to volkensiflavone hexamethyl ether (6) led tolower activity and decreased cytotoxicity.

[0117] All of these biflavanoids were relatively inactive towardparainfluenza type 3, respiratory syncytial, measles, and adenovirustype 5 viruses, as shown in Table 8 and Table 9, except amentoflavone(1) and rhusflavanone (9) which exhibited some slight activity againstrespiratory syncytial virus and measles virus, respectively. TABLE 8Measles Virus Adenovirus Type 5 CPI Inhibition Neutral Red CPEInhibition Neutral Red EC₅₀*¹ IC₅₀*² EC₅₀*¹ IC₅₀*² EC₅₀*¹ IC₅₀*² EC₅₀*¹IC₅₀*² Sample ug/ml ug/ml SI*³ ug/ml ug/ml SI*³ ug/ml ug/ml SI*³ ug/mlug/ml SI*³ Ribavirin* 3 150 50 1 150 150 — — — — — — HPMPA* — — — — — —30 80 3 8 40 5 Amentoflavone (1) <40 <10 0 <20 <60 3 >100 18 0 >100 74 0Agathisflavone (2) <60 <10 0 <10 <30 3 15 18 1 22 37 1 Robustaflavone(3) <14 <14 1 −30 ˜100 1 >100 56 0 >100 102 0 Hinokiflavone (4) <3 <4 1<5 <11 2 >10 22 0 19 27 1 Volkensiflavone (5) <12 <10 1 <6 <10 1 56 471 >32 30 0 Volkensiflavone <70 <60 1 <7 <13 1 >100 47 0 >100 50 0hexamethyl ether (6) Rhusflavanone (9) 14 21 2 5 40 8 56 47 1 33 15 0Rhusflavanone ˜3 <4 0 <10 <10 0 >10 22 0 19 26 1 hexaacetate (10)Succedaneaflavone (11) ˜32 <23 0 <12 <20 1 10 22 0 19 27 1Succedaneaflavone ˜3.2 <4 0 <2 <200 <1 20 19 1 6 6 1 hexaacetate (12)

[0118] TABLE 9 Parainfluenza Type 3 Virus Respiratory Syncytial VirusCPI Inhibition Neutral Red CPE Inhibition Neutral Red EC₅₀*¹ IC₅₀*²EC₅₀*¹ IC₅₀*² EC₅₀*¹ IC₅₀*² EC₅₀*¹ IC₅₀*² Sample ug/ml ug/ml SI*³ ug/mlug/ml SI*³ ug/ml ug/ml SI*³ ug/ml ug/ml SI*³ Ribavirin* 25 245 10 17 33119 12 120 10 6 60 10 Amentoflavone (1) >100 ˜56 0 ˜32 ˜56 2 ˜10 ˜56 6˜21 ˜35 2 Agathisflavone (2) >100 ˜33 0 >100 ˜34 0 >10 ˜8 0 ˜24 ˜15 0Robustaflavone (3) <100 56 0 39 79 2 >100 ˜56 0 ˜50 ˜100 2 Hinokiflavone(4) <1 1 0 5 7 1 >3.2 2.5 0 >3.2 6 0 Volkensiflavone (5) 47 50 1 15 44 332 56 2 28 13 0 Volkensiflavone <32 ˜15 0 >32 ˜33 0 ˜18 ˜30 2 >32 ˜59 >1hexamethyl ether (6) Rhusflavanone (9) >100 47 0 85 30 0 >10 18 0 27 200 Rhusflavanone >10 22 0 32 19 0 >10 13 0 19 32 2 hexaacetate (10)Succedaneaflavone (11) >10 ˜22 0 23 ˜29 0 >10 ˜13 0 ˜16 ˜10 0Succedaneaflavone >10 ˜14 0 >32 ˜13 0 >3 4 0 6 6 1 hexaacetate (12)

EXAMPLE 9 Anti-HIV Viral Activity of Biflavanoids

[0119] We have investigated the anti-HIV-1 RT activity of biflavanoidsisolated from Rhus succedanea, amentoflavone (1), agathisflavone (2),robustaflavone (3), hinokiflavone (4), rhusflavanone (9),succedaneaflavanone (11), and from Garcinia multiflora, volkensiflavone(5), morelloflavone (7), GB-1a (13), GB-1a 7″-O-b-glucoside (15), GB-2a(16), and their sulfate potassium salt, methyl ether, and acetylderivative, volkensiflavone hexaacetate (6), morelloflavone heptamethylether (8), rhusflavanone hexaacetate (10), succedaneaflavanonehexaacetate (12), GB-1a hexamethyl ether (14), and robustaflavonetetrasulfate potassium salt (17).

[0120] Anti-HIV-1 RT Assay. The HIV-1 RT is a 66-kDa recombinant enzymeobtained from an Escherichia coli expression system using a geneticallyengineered plasmid; the enzyme was purified to near homogeneity.Synthetic DNA segments were used to introduce initiation and terminationcodons into the HIV-1 RT coding sequence, which permits expression oflarge quantities of HIV-1 RT in E. coli. The enzyme was shown to beactive in RT assays and was inhibited by several known antiretroviralagents (e.g. AZT and suramin). The purified recombinant enzyme wassufficiently similar to the viral enzyme that it can be substituted forthe latter in drug screening assays. The recombinant HIV-1 RTpreparation used in all experiments had a protein concentration of 0.11ug/mL and an activity of 238 nmol TTP incorporated per 10 min per mg ofprotein at 37° C. Prior to performing an experiment, the enzyme wasdiluted tenfold with buffer analogous to that used in the assay.

[0121] The assay mixture (final volume 100 uL) contained the following:50 mM Tris-HCl buffer (pH 8.0), 150 mM KCl, 5 mM MgCl, 0.5 mM ethyleneglycol-bis-(b-aminoethylether)-N,N′-tetraacetic acid (EGTA), 5 mMdithiothreitol 0.3 mM glutathione, 2.5 mg/mL bovine serum albumin, 41 mMpoly A [S260 (mM)=7.8], 9.5 mM oligo (dT)12-18 [S265(mM)=5.6], 0.05%Triton X-100, 20 mM TTP, and 0.5 mCi of [³H]TTP. The reaction wasstarted by the addition of 10 uL of HIV-1 RT, and the mixture waspermitted to incubate at 37° C. for 1 h. Reactions were terminated bythe addition of 25 uL of 0.1 M EGTA followed by chilling on ice.Aliquots of each reaction mixture (100 uL) were then spotted uniformlyonto circular 2.5 cm DE-81 (Whatman) filters, kept at ambienttemperature for 15 minutes, and washed four times with 5% aqueousNa₂HPO₄7H₂O. This was followed by two more washings with doublydistilled H₂O. Finally, the filters were thoroughly dried and subjectedto scintillation counting in a nonaqueous scintillation fluid.

[0122] For testing enzyme inhibition, five serial dilutions of samplesin DMSO (10 uL) were added to the reaction mixtures prior to theaddition of enzyme (10 uL). The final DMSO concentration used was 10%.The highest concentration of pure natural products and plant extractstested was 200 μg/mL. Control assays are performed without the compoundsor extracts, but an equivalent volume of DMSO was added. Fargaroninechloride was used as the positive control substance. This compound wasisolated from Fagara xanthoxyloides Lam. Other positive controlsubstances used were suramin (IC₅₀ 18 μg/mL) and daunomycin (IC₅₀ 125μg/mL). The assay procedure and the concentration of all components werethe same as that described above.⁴⁷

[0123] Anti-HIV-1 RT Assay in Primary Human Lymphocytes

[0124] Cell Culture. Human PBM cells from healthy HIV-1 seronegative andhepatitis B virus seronegative donors were isolated by Ficoll-Hypaquediscontinuous gradient centrifugation at 1,000 x g for 30 min, washedtwice with phosphate-buffered saline (pH 7.2, PBS), and pelleted bycentrifugation at 300 x g for 10 min. Before infection, the cells werestimulated by phytohemagglutinin (PHA) at a concentration of 6 μg/mL for2-3 days in RPMI 1640 medium, supplemented with 15% heat-inactivatedfetal calf serum, 1.5 mM L-glutamine, penicillin (100 U/mL),streptomycin (100 μg/mL), and 4 mM sodium bicarbonate buffer.

[0125] Viruses. HIV-1 (strain LAV-1) was obtained from Dr. P. Feorino(Emory University, Atlanta, Ga.). The virus was propagated in human PBMcells using RPMI 1640 medium, as described previously⁵⁸ without PHA orfungizone and supplemented with 26 units/mL of recombinant interleukin-2(Cetus Corporation, Emeryville, Calif.) and 7 μg/mL DEAE-dextran(Pharmacia, Uppsala, Sweden). Virus was obtained from cell-free culturesupernatant and was titrated and stored in aliquots at −70° C. untiluse.

[0126] Inhibition of Virus Replication in Human PBM Cells. UninfectedPHA-stimulated human PBM cells were infected in bulk with a suitabledilution of virus. The mean reverse transcriptase (RT) activity of theinocula was about 60,000 dpm RT activity/106 cells/10 mL. Thisrepresents, by a limiting dilution method in PBM cells, a multiplicityof infection of about 0.01. After 1 h, the cells were uniformlydistributed among 25 cm² flasks to give a 5 mL suspension containingabout 2×10⁶ cells/mL each. The samples at twice their finalconcentration in 5 mL of RPMI 1640 medium, supplemented as describedabove, were added to the cultures. The cultures were maintained in ahumidified 5% CO₂-95% air incubator at 37° C. for six days afterinjection, at which point all cultures were sampled for supematant RTactivity. Previous studies had indicated that maximum RT levels wereobtained at that time.

[0127] RT Activity Assay. A volume of supernatant (1 mL) from eachculture was clarified of cells at 300 x g for 10 min. Virus particleswere pelleted at 12,000 rpm for 2 h using a Jouan refrigeratedmicrocentrifuge (Model MR 1822) and suspended in 100 μL of virusdisrupting buffer (50 mM Tris-HCl, pH 7.8, 800 mM NaCl, 20% glycerol,0.5 mM phenylmethyl sulfonyl fluoride, and 0.5% Triton X-100).

[0128] The RT assay was performed in 96-well microtiter plates, asdescribed by Spira.⁶⁹ The reaction mixture, which contained 50 mMTris-HCl, pH 7.8, 9 mM MgCl₂, 5 mM dithiothreitol, 4.7 μg/mL(rA)n(dT)12-18, 140 μM dAPT, and 0.22 μM [³H]TTP (specific activity 78.0Ci/mmol, equivalent to 17,300 cpm/pmol; NEN Research Products, Boston,Mass.), was added to each well. The sample (20 μL) was added to thereaction mixture, which was then incubated at 37° C. for 2 h. Thereaction was terminated by the addition of 100 μL of 10% trichloroaceticacid (TCA) containing 0.45 mM sodium pyrophosphate. The acid-insolublenucleic acids which precipitated were collected on glass filters using aSkatron semi-automatic harvester (setting 9). The filters were washedwith 5% TCA and 70% ethanol, dried and placed in scintillation vials.Scintillation fluid (Ecolite, ICN, Irvine, Calif.) (4 mL) was added andthe amount of radioactivity in each sample was determined using aBeckman liquid scintillation analyzer (Model LS 3801). The results wereexpressed in dpm/mL of original clarified supernatant. The proceduresfor the anti-HIV assays in PBM cells described above have beenpublished.^(67,69)

[0129] Cytotoxicity Studies in PBM Cells. The compounds were evaluatedfor their potential toxic effects on uninfected PHA-stimulated human PBMcells. The cells were cultured with and without drug for 24 h, at whichtime radiolabeled thymidine was added. The assay was performed asdescribed previously.³⁵ Alternately, cells are counted on day 6 using ahemacytometer and/or Coulter counter as described previously.⁶⁸

[0130] Median-Effect Method. EC₅₀ and IC₅₀ values were obtained byanalysis of the data using the median-effect equation.⁴² These valueswere derived from the computer-generated median effect plot of thedose-effect data using a commercially available program.⁴³

[0131] The results shown in Table 10 indicate that both hinokiflavone(4) and robustaflavone (3) demonstrated similar activity against HIV-1RT at an IC₅₀ (50% inhibition dose) of 35.2 μg/mL and 33.7 μg/mL,respectively. The water soluble form of robustaflavone, robustaflavonetetrasulfate K-salt (17) exhibited 95.5% inhibition at a concentrationof 200 ug/mL, with an IC₅₀ value of 144.4 ug/nL. Amentoflavone (1),agathisflavone (2), morelloflavone (7), GB-1a (13), and GB-2a (16) weremoderately active against HIV-1 RT with IC₅₀ values of 64.0 μg/mL, 53.8μg/mL, 64.7 μg/mL, 127.8 μg/mL, and 94.6 μg/mL, respectively. The otherbiflavanoids were either slightly active or inactive against HIV-1 RT.

[0132] The results of both studies are presented in Table 10. Theresults of the inhibitory activity tests using HIV-1 RT enzyme (p66/p51heterodimer) indicated that the biflavones, two apigenin units linkedeither with C—C or C—O—C bonds, exhibited significant activity.Robustaflavone (3) (two apigenins linked through an I-6-II-3′ linkage)and hinokiflavone (4) (I-6-O-II-4′ linkage) demonstrated similaractivity, with 50% inhibition (IC₅₀) at doses of 35.2 μg/mL and 33.7μg/mL, respectively. The IC₅₀ values of amentoflavone (1) (I-8-II-3′linkage) and agathisflavone (2) (I-6-II-8 linkage) were 64.0 μg/mLand 53.8 μg/mL, respectively. TABLE 10 Anti-HIV-1 RT Activity ofBiflavanoids Anti-HIV-1 RT Ant-HIV-1 Cytotoxicity % Inhibition in PBM inPBM Selective at 200 IC₅₀ cells cells Index Compounds ug/ml (uM) EC₅₀(uM) IC₅₀ (uM) (SI) Apigenin 72 (443) Naringenin 34.9 Weakly ActiveAmentoflavone (1) 97.3 (119) >10.94 35 Agathisflavone (2) 99.8 (100)7.3, 6.0 25 <1 Robustaflavone (3) 91.4  (65) >100 77 <1 Hinokiflavone(4) 89.0  (62) 4.1 9.1 <1 Volkensiflavone (5) 45.3 Weakly Active 2.2Volkensiflavone 0.00 Inactive Me₂ (6) Morelloflavone (7) 99.2 (116) 5.7,8.0 82 12 Rhusflavanone (9) 14.1 Inactive Rhusflavanone Ac₆ (10) 0.00Inactive Succedanea- 22.1 Inactive flavanone (11) Succedanea- 0.00Inactive flavanone Ac₆ (12) GB-1a (13) 86.0 (236) 38.0 88 2.3 GB-1a Me₆(14) 0.00 Inactive GB-1a glucoside (15) 1.46 Inactive GB-2a (16) 96(170) Robustaflavone 95.5 144.4 tetrasulfate K-salt

[0133] Biflavanoids constructed of flavanone-flavone units throughI-3-II-8 linkages were moderately to weakly active, i.e. morelloflavone(7) (naringenin I-3-II-8 quercetin) demonstrated moderate activity, withan IC₅₀ value of 64.7 μg/mL, while volkensiflavone (5)(narnigeninI-3-II-8 apigenin) was weakly active. Biflavanones consisting of twonaringenin units or naringenin-eriodictol through I-3-II-8 linkagesexhibited moderate activity, such as GB-1a (13) (IC₅₀ 127.8 μg/ML) andGB-2a (16) (IC₅₀ 94.6 μg/mL). Biflavanones such as rhusflavanone (9) andsuccedaneaflavanone (11), comprised of two naringenin units linkedthrough either I-6-II-8 or I-6-II-6 linkages, were completely inactive.

[0134] Other structural characteristics were related to activity in ourstudy. Methylation of the hydroxyl groups of the biflavanoids resultedin diminished activity. For instance, morelloflavone heptamethyl ether(8), volkensiflavone hexamethyl ether (6), and GB-1a hexamethyl ether(14), were inactive; all had exhibited moderate activity beforealkylation. The fact that GB-1a-7″-O-glucoside (15), demonstrated noactivity indicated that the 7″-hydroxyl group was especially importantfor anti-HIV-1 RT activity.

[0135] Six biflavanoids that were determined to be active in the HIV-1RT enzyme assay were tested in human PBM cells infected with HIV-1(strain LAV). These results are presented in Table 10. It has beenobserved that, although robustaflavone (3) exhibited significantinhibitory activity in the HIV-1 RT enzyme assay, it was found to beinactive in the assay for the PBM cells infected with HIV-1. Howevermorelloflavone (7), in the whole cell assay, exhibited potent inhibitoryactivity with an EC₅₀ (50% effective dose) value of 5.7 (8.0) μg/mL.Morelloflavone only possessed moderate activity in the anti-HIV-1 RTassay (IC₅₀ 64.7 μg/mL; 116.3 μM). This may suggest that the activity ofthese biflavanoids may be dependent upon different cellular mechanisms.

[0136] Other active compounds were hinokiflavone (4) and GB-1a (13),which exhibited good activity inhibiting viral replication in human PBMcells, but also high toxicity against uninfected PHA-stimulated humanPBM cells. The other compounds (amentoflavone (1) and agathisflavone(2)) assayed in PBM cells appeared to either lack antiviral potency ordisplay poor selectivity. From these results, it was concluded thatbiflavanoids comprised of flavanone (naringenin) and flavone (luteolin)via a I-3-II-8 bond demonstrate the most promising anti-HIV-1 activity.

[0137] In the past, some monoflavonoids have been reported todemonstrate anti-HIV activity. Baicalein (5,6,7-trihydroxyflavone),tiliroside (kaempferol 3-beta-D (6″-p-coumaroyl)glucoside), quercetin(3,3′,4′,5,7-pentahydroxyflavone), kaempferol(3,4′,5,7-tetrahydroxyflavone), and quercetagetin(3,3′,4′,5,6,7-hexahydroxyflavone) exhibited inhibitory activity againstHIV-1 reverse transcriptase, whereas luteolin(3′,4′,5,7-tetrahydroxyflavone) and apigenin (4′,5,7-trihydroxyflavone)showed moderate to slight inhibition, and naringenin(4′,5,7-trihydroxyflavanone) was completely inactive.^(63,64,70) Thisrevealed that the presence of both the unsaturated double bond betweenpositions 2 and 3 of the flavonoid pyrone ring (e.g. flavone), andeither the 3 hydroxyl groups introduced at the 5, 6, and 7 positions(bicalein) or the 3, 3′, and 4′ positions (quercetin) were aprerequisite for inhibition of RT activity.

[0138] In our study, apigenin exhibited moderate activity and naringenindemonstrated slight inhibition. Biflavanoids which consisted of twoapigenin units (amentoflavone (1), agathisflavone (2), robustaflavone(3), and hinokiflavone (4)) demonstrated significant activity.Biflavanoids constructed of flavanone and flavone units (morelloflavone(7)) and biflavanone, linked through I-3-II-8 (GB-1a (13) and GB-2a(16)) were moderately active, and biflavanones linked through ring A oftwo naringenin units (rhusflavanone (9) and succedaneaflavanone (11))were inactive. This structure-activity comparison again demonstratesthat hydroxyl groups and at least one flavone unit in the biflavanoidsare required for activity. A I-3-II-8 linkage is also necessary forbiflavanones to exhibit activity. A firther conclusion is thatpreviously active compounds become inactive when hydroxy groups aremethylated.

EXAMPLE 10 Anti-Herpes Viral Activity of Biflavanoids

[0139] Anti-Herpes Viral Assay: The viruses used in the primary screenfor anti-viral activity against herpes viruses consisted of: HerpesVirus 1 (HSV-1 E-377 strain), Herpes Virus 2 (HSV-2 MS strain),Cytomegalovirus (HCMV AD 169 strain), Varicella Zoster Virus (VZV EllenStrain), and Epstein-Barr Virus (EBV), superinfection of Raji or Daudicells with P3HR-1.

[0140] The assay for the inhibition of the cytopathic effect (CPE) forHSV, HCMV and VZV was as follows: Low passage human foreskin fibroblastcells were seeded in 96-well tissue culture plates 24 h prior to use, ata cell concentration of 2.5×10⁴ cells/mL in 0.1 L of minimal essentialmedium (MEM) supplemented with 10% fetal bovine serum (FBS). The cellswere then incubated for 24 h at 37° C. in a CO₂ incubator. Afterincubation, the medium was removed and 100 mL of MEM containing 2% FBSwas added to all but the first row. In the first row, 125 mL of the testcompound was added in triplicate wells. Medium alone was added to bothcell and virus control wells. The test compound in the first row wasdiluted serially 1:5 throughout the remaining wells by transferring 25mL using a Cetus Liquid Handling Machine. After dilution of thecompound, 100 mL of the appropriate virus concentration was added toeach well, excluding cell control wells which received 100 mL of MEM.For HSV-1 and HSV-2 assays, the virus concentration utilized was 1000PFUs per well. For CMV and VZV assays, the virus concentration added was2500 PFUs per well. The plates were then incubated at 37° C. in a CO₂incubator for three days for HSV-1 and HSV-2, 10 days for VZV, or 14days for CMV. After the incubation period, the media was aspirated andthe cells stained with a 0.1% crystal violet solution for 30 min. Thestain was then removed and the plates rinsed using tap water until allthe excess stain was removed. The plates were allowed to dry for 24 hand then read on a Skatron Plate reader at 620 nm.

[0141] VZV Plaque Reduction Assay. Two days prior to use, HFF cells wereplated into six-well plates and incubated at 37° C., with 5% CO₂atmosphere and 90% humidity. On the date of assay, the test compound wasmade up at twice the desired concentration in 2X MEM using sixconcentrations of the compound. The initial starting concentrations wereusually from 200 ug/mL to 0.06 ug/mL. The VZV was diluted in 2X MEMcontaining 10% FBS to a desired concentration which would give 20-30plaques per well. The media was then aspirated from the wells and 0.2 mLof the virus was added to each well in duplicate, with 0.2 mL of mediabeing added to the drug toxicity wells. The plates were then incubatedfor 1 h with shaking every 15 min. After the incubation period, meanequal amount of 1% agarose was added to an equal volume of each testcompound dilution. This provided final test compound concentrationsbeginning with 100 ug/mL and ending with 0.03 ug/mL, and a final agaroseoverlay concentration of 0.5%. The test compound agarose mixture wasapplied to each well in 2 mL volumes. The plates were then incubated,the stain aspirated, and plaques counted using a stereomicroscope at 10xmagnification for ten days, after which the cells were stained with a1.5% solution of neutral red dye. On days three and six an additional 1mL overlay with equal amounts of 2X MEM and 1% agarose were added. Atthe end of the 4-6 h incubation period, the stain was aspirated andplaques counted using a stereomicroscope at 10x magnification.

Herpes viruses (HSV-1, HSV-2, HCMV, VZV, and EBV)

[0142] The results of the anti-herpes viruses activity assays of thesebiflavanoids are presented in Table 11. Among the compounds studied,only robustaflavone (3) exhibited significant inhibitory activitiesagainst HSV-1 and HSV-2 viruses. Activity values are measured byeffective concentration (EC₅₀) and cytotoxicity concentration (CC₅₀) atwhich 50% of cells are free from pathogens or 50% of cells die. Thevalues for robustaflavone (3) are an EC₅₀ of 8.6 μg/mL and CC₅₀>100μg/mL, which results in a selectivity index of >11.6. The anti-viralactivity of robustaflavone (3) against HSV-2 produced an EC₅₀ value of8.5 μg/mL, a CC₅₀ of >100 μg/mL, and a SI of >11.8. Other resultsinclude amentoflavone (1) which demonstrated only slight activityagainst HSV-1. Volkensiflavone (5) exhibited weak inhibitory activityagainst both HCMV and VZV. Methylation of volkensiflavone (5) intovolkensiflavone hexamethyl ether (6), resulted in the loss of activity,and a decrease in toxicity against HCMV, but an increase in activity andtoxicity against VZV. Acetylation of rhusflavanone (9) to rhusflavanonehexaacetate (10) increased the activity and toxicity against HSV-1 andHSV-2. Acetylation of succedaneaflavanone (11) into succedaneaflavanonehexaacetate (12) led to a slight decrease of both activity and toxicity,and resulted in almost equal SI values. When assayed for activityagainst VZV, the acetylation product (12) resulted in an SI value whichincreased from <3 to 9.6. TABLE 11 HSV-1 (HFF Cells) HSV-2 (HFF Cells)HCMV (HFF Cells) VZV (HFF Cells) CPE Inhibition CPE Inhibition CPEInhibition Plaque Reduction EC₅₀*¹ CC₅₀*² EC₅₀*¹ CC₅₀*² EC₅₀*¹ CC₅₀*²EC₅₀*¹ CC₅₀*² Sample ug/ml ug/ml SI*³ ug/ml ug/ml SI*³ ug/ml ug/ml SI*³ug/ml ug/ml SI*³ ACT* 1.5 — 0.9 — — — — — 0.5 — — GVC* — >100 — — — 0.4— — — — — Amentoflavone (1) 17.9 >100 >5.6 48.0 >100 >2.150.8 >100 >1.9 >4.0 9.3 >2.3 Agathisflavone (2) >100 >100 0 >100 >100 094.8 >100 >1.0 >4 12.0 <3.0 Robustaflavone (3) 8.6 >100 >11.68.5 >100 >11.8 54.8 >100 >1.8 — — — Hinokiflavone (4) >20 77.7 <3.9 >2077.5 <3/9 >0.8 2.6 <3.2 >4.0 16.8 <4.2 Volkensiflavone (5) >100 >100 087.9 >100 >1.1 >4.0 16.8 <4.2 >20 80.0 <4.0 Volkensiflavone >100 >1000 >100 >100 0 >100 >100 0 3.3 11.1 3.4 hexamethyl ether (6)Rhusflavanone (9) >100 >100 0 15.7 >100 >6.4 >4.0 13.7 <3.4 >4 16.0 <4Rhusflavanone >4.0 18.5 <4.6 >4.0 18.5 <4.6 >4.0 13.4 <3.3 11.3 46.7 4.1hexaacetate (10) Succedaneaflavone (11) >20 60.7 <3.0 >20 60.7 <3.0 >2055.5 <2.7 >20 60.0 <3.0 Succedaneaflavone >4 17.7 <4.4 >4.0 17.7<4.4 >4.0 14.4 <3.5 7.1 68.0 9.6 hexaacetate (12)

EXAMPLE 11 In Vivo Evaluation of Robustaflavone in a Murine InfluenzaModel

[0143] In this Example, a series of in vivo experiments were run todetermine if robustaflavone is efficacious against an experimentallyinduced influenza virus infection in mice. Prior to beginning thisstudy, a series of preliminary experiments were run to determine themaximum tolerated dose of this compound in mice. Since the compound isnot soluble in aqueous medium, it was suspended in 0.4%carboxymethylcellulose (CMC), a vehicle commonly used forwater-insoluble compounds. When it was found that the compound was welltolerated at high dosages in this suspension, the question arose as towhether it was being adequately absorbed by the animal. Some studieswere thus conducted using other vehicles in which the compound was moresoluble. These vehicles included dimethylsulfoxide (DMSO), dimethylformamide (DMF), and polyethylene glycol (PEG).

Materials and Methods

[0144] Animals: Female 13-15 g specific pathogen-free BALB/c mice wereobtained form Simonsen Laboratories (Gilroy, Calif.). They werequarantined 24 h prior to use, and maintained on Wayne Lab Blox and tapwater. After being infected, their drinking water contained 0.006%oxytetracycline (Pfizer, New York, N.Y.) to control possible secondarybacterial infections.

[0145] Virus: A/NWS/33 (H1N1) was obtained from K. W. Cochran, Univ. ofMichigan (Ann Arbor, Mich.). A virus pool was prepared in MDCK cells;this was titrated in mice, ampuled, and stored at −80° C. until used.

[0146] Compounds: Robustaflavone was stored at room temperature untilused. Ribavirin, used as a positive control, was obtained from ICNPharmaceuticals (Costa Mesa, Calif.). Vehicles considered included DMSO(Sigma Chemical Co., St. Louis, Mo.), DMF (Sigma), PEG M.W. 200 (AldrichChemical Co. Milwaukee, Wis.), 0.4% CMC (Sigma) and1-methyl-2-pyrrolidinone (MPD, Aldrich).

[0147] Arterial Oxygen Saturation (SaO₂) Determinations: SaO₂ wasdetermined using the Ohmeda Blox 3740 pulse oximeter (Ohmeda,Louisville, Ohio)). The ear probe attachment was used, the probe placedon the thigh of the animal, with the slow instrument mode selected.Readings were made after a 30 second stabilization time on each animal.Use of this device for measuring effects of influenza virus on arterialoxygen saturation have been described.⁷²

[0148] Lung Virus Determinations: Each mouse lung was homogenized andvarying dilutions assayed in triplicate for infectious virus in MDCKcells as described previously.⁷³

Experiment Design

[0149] 1. Toxicity Determination of robustaflavone in CMC Vehicle: Thecompound was suspended in 0.4% CMC at a concentration of 37.5 mg/mL tomake a dosage of 500 mg/kg/day. It was injected i.p. into 2 mice dailyfor 5 days. The mice were weighed and deaths noted daily.

[0150] 2. Toxicity Determination of robustaflavone in 100% DMSO: Thecompound was dissolved in DMSO at a concentration of 25 mg/mL and in alater experiment in a concentration of 11.25 mg/mL to make dosages of250 and 75 mg/kg/day, respectively. The higher dosage was injected i.p.into mice twice daily for 5 days in a volume of 0.1 mL/injection dailyfor 5 days in a volume of 0.05 mL/injection. As controls, mice weretreated by the same treatment schedule with DMSO only in volumes of 0.1or 0.05 mL/injection. Weight gain and mortality was determined in theseanimals.

[0151] 3. 3. Toxicity Determination of DMF and PEG only: DMF and PEG 200in a concentration of 100% were injected i.p. into separate groups ofmice daily for 5 days using a volume of 0.05 mL/injection. Again,effects on host weight and deaths of mice were monitored.

[0152] 4. Effect of robustaflavone in CMC or in DMSO on influenza virusinfection in mice. In the study with CMC, robustaflavone was used indosages of 200 and 100 mg/kg/day; using DMSO vehicle, the dosages were75 and 37.5 mg/kg/day, with the compound administered i.p. twice dailyfor 5 days beginning 4 h pre-virus exposure. The mice were used in eachdose to monitor effects on SaO₂ and death; from an additional group ofsimilarly infected and treated mice, 3 animals were killed on days 3, 5,7 and 9 to assay for lung score (0=normal, 4=maximal consolidation),weight, and virus titer. Three to four mice were used as toxicitycontrols, which were weighed prior to treatment and again 18 h aftertreatment termination, and deaths noted daily. Ribavirin, dissolved insaline, was used in a dose of 75 mg/kg/day with the same treatmentschedule. Three sets of virus controls were used: Infected-untreated,infected-treated with CMC only, and infected-treated with DMSO only.Twenty animals were used in each of these control groups to monitor SaO₂and death, with 3 additional mice taken in parallel with treated animalsto determine effects on lung consolidation and virus titer. Two sets ofnormal controls were used; one group of three mice was weighed and heldin parallel with the toxicity controls. From the second group three micewere killed on days 3 and 9 for comparison of lung score and weight.

[0153] Statistical Evaluation: Increase in survivor number was evaluatedusing chi square analysis with Yates' correction. Mean survival timeincreases, virus titer and SaO₂ value differences were analyzed byt-test. Lung consolidation scores were evaluated by ranked sum analysis.

Results and Discussion

[0154] Toxicological Effects on Various Vehicles: The results of thevarious experiments with the vehicles considered are summarized in Table12. CMC was the most well tolerated, followed by DMSO. DMF and PEG 200were lethally toxic to the mice. One mouse died immediately followingthe day 4 i.p. treatment with DMSO; since this animal died instantly itis probable the death was due to penetration of an organ by the needleas it was administered into the peritoneal cavity. Using the 0.05 mLvolume of DMSO, the animals appeared to tolerate this vehicle betterthan at 0.1 mL. DMF was highly lethal, killing both animals after twoinjections, and PEG 200 was only slightly better, with all mice dyingafter 3 injections.

[0155] Based on the above data, both CMC and DMSO were used as solventsfor robustaflavone, the latter used in injection volumes of 0.05 mL.

[0156] Dose Range-Finding Studies with Robustaflavone in Mice: Using CMCas vehicle, robustaflavone appeared to be quite insoluble, with denseyellow particulate material seen in the formulation. When injected i.p.twice daily for 5 days, a dose of 200 mg/kg/day appeared reasonably welltolerated, the treated animals surviving therapy but losing 0.1 g ofweight in the 5-day treatment period. The material was very soluble inDMSO, forming a clear solution. A 250 mg/kg/day dose injected i.p. twicedaily for 5 days was lethally toxic to the mice, all animals dying byday 5 of treatment and a 6 g weight loss seen. The injection volume inthis experiment was 0.1 mL, when the experiment was repeated using 0.05mL injection volume, the dosage was lowered to 75 mg/kg/day. At thisdose, all mice survived, although they lost 2 g of weight during the5-day treatment period.

[0157] The data using CMC as vehicle suggests the compound was not beingwell absorbed in the animal, so for the antiviral experiment it wasdecided to use doses of 200 and 100 mg/kg/day. The DMSO studiesindicated 75 mg/kg/day may be approaching the maximum tolerated dose, sothat dose and 37.5 mg/kg/day were chosen for the in vivo antiviralexperiment.

[0158] Effect of robustaflavone in DMSO on Influenza A Virus Infectionsin Mice: The results of this experiment are summarized in Table 14 andFIG. 1 through 4. It was found that the 75 mg/kg/day dose in thisantiviral experiment was lethally toxic to the mice; the 37.5 mg/kg/daydose killed 2 of 3 toxicity control mice as well. Due to this apparenttoxicity, the effects on survivors and SaO₂ values were inconclusive.This excess toxicity did not correlate with the earlier-runrange-finding study, although in the latter study marked weight loss wasseen suggesting the compound was approaching a lethally toxic dose.

[0159] A review of FIG. 2 and 3, showing effects of treatment on lungscores and lung weights, indicates a significant effect of this compoundon lowering lung scores and weights. This effect was dose-responsive,and suggests robustaflavone may have a significant influenza-inhibitoryeffect which may also be seen at a dose more well tolerated to the mice.

[0160] DMSO used alone was not lethal to the mice, but infected animalstreated with DMSO only died approximately 2 days sooner than untreatedinfected controls (Table 12). This suggests the DMSO injection mayresult in an enhancement of the infection.

[0161] Ribavirin, run in parallel as a positive control, was highlyactive in inhibiting the infection using all evaluation parameters.

[0162] Effect of robustaflavone in CMC on influenza A virus infectionsin mice: The results of this study are seen in Table 14 and in FIGS. 5through 8. Robustaflavone appeared to be well tolerated in thisexperiment, with all toxicity controls surviving and host weight gainapproaching that seen with normal controls run in parallel observed. Thetherapy did not prevent death, but did increase mean survival times in adose-responsive manner. SaO₂ levels remained high in these treatedanimals as well (Table 14, FIG. 5).

[0163] Treatment with this compound also inhibited lung consolidation ina dose-responsive fashion as seen in FIGS. 6 and 7.

[0164] These data indicate that: 1) Robustaflavone can be inhibitory tothe in vivo influenza infection and 2) there is apparently at least apartial absorption of the compound since the dose-responsive effectswere seen.

[0165] It may be pertinent to note that two flavones have previouslybeen reported to have influenza virus-inhibitory effects.5,7,8,4′-Tetrahydroxyflavone was reported in 1992⁷⁴ to prevent viralproliferation in lungs of infected mice when the compound wasadministered either by the intranasal or oral routes. The related8-methylether compound, 5,7,4′-trihydroxy-8-methoxyflavone, wassimilarly effective when administered intranasally or by the i.p.routes.⁷⁴⁻⁷⁷ Research by these investigators indicated the compoundsreduce viral replication by inhibiting fusion of the virus withendosome/lysome membrane which occurs at an early stage of the virusinfection cycle and may also inhibit budding of the progeny virus fromthe cells surface.^(77,78) The vehicle for these flavones wasNa₂CO₃/saline.

Conclusion

[0166] Robustaflavone was evaluated against influenza A/NWS/33 (H1N1)virus infections in mice using two vehicles, 0.4% carboxymethylcellulose(CMC) and 100% dimethylsulfoxide (DMSO) Treatment was i.p. twice dailyfor 5 days beginning 4 h pre-virus exposure. The compound in DMSO wastoxic to the mice at the two dosages employed, 75 and 37.5 mg/kg/day;despite this toxicity, significant reduction in lung consolidation wasseen. When used in CMC, the doses of 200 and 100 mg/kg/day used werewell tolerated and both inhibited lung consolidation and slowed the meanday to death of the animals. TABLE 12 Toxicological Effects of CMC,DMSO, DMSF, and PEG 200 in BALB/c Mice¹ Volume/ Mean Host InjectionSurv/ Mean Day Wt. Vehicle (ml) Total To Death Change (g)² 0.4% 0.1 33 >21 1.7 Carboxymethyl- cellulose (CMC) 100% DMSO 0.1 2 2 >21 −.26 100%DMSO 0.05 1 2 4.0 −0.3 100 MF 0.05 0 2 1.0 7 100% PEC 200 0.08 1 2 5.0−2.2 100% PEC 200 0.1 0 2 2.0 −2.8

[0167] TABLE 13 Effect of i.p. Treatment with Robustaflavone in DMSOVehicle on influenza A (H1N1) Virus Infections in Mice Animals: 13-15 gfemale BALB/c Mice Treatment Schedule: bid × 5 beg −4 Virus: Influenza A(A/NWS/33 (H1N1), h pre-virus exposure i.n. Treatment route: i.p. DrugDiluent: Robustaflavone 0.4% Experiment Duration: 21 days DMSO;Ribavirin Saline Toxicity Controls Infected, Treated Dosage Surv/ MeanWeight Surv/ MST^(b) Mean Compound (mg/kg/day) Total Change (g)^(a)Total (days) SaO₂ ^(c) (%) Robustaflavone 75 0/3 −1.7 0/9  3.2 70.6 37.51/3 −0.8 0/10 8.0 73.8 Ribavirin 75 3/3 −0.5  10/10** >21.0** 87.1**DMSO — — — 0/20 9.6 82.6 Untreated — — — 0/20 11.4 84.2 Normals — 3/3 2.0 — — 87.9

[0168] TABLE 14 Effect of i.p. Treatment with Robustaflavone in CMCVehicle on influenza A (H1N1) Virus Infections in Mice Animals: 13-15 gfemale BALB/c Mice Treatment Schedule: bid × 5 beg Virus: Influenza A(A/NWS/33 (H1N1), -4 h pre-virus exposure i.n. Treatment route: i.p.Drug Diluent: Robustaflavone 0.4% Experiment Duration: 21 days CMC;Ribavirin Saline Toxicity Infected, Controls Treated Mean Weight Com-Dosage Surv/ Change Surv MST^(b) Mean pound (mg/kg/day) Total (g)^(a)Total (days) SaO₂ ^(c) (%) Robusta- 200  3/3 1.5  0/9 11.1 84.6**flavone 1005  4/4 1.9  0/10 9.8 85.1** Ribavirin 75 3/3 −0.5  10/10** >21.0** 87.1** CMC — — —  0/16 9.3 80.4 Untreated — — —  0/2011.4  84.2 Normals — 3/3 2.0 — — 87.9

CONCLUSION

[0169] Robustaflavone, a naturally occurring biflavanoid isolated fromthe seed kernel extract of Rhus succedanea, was found to be a potent invitro inhibitor of hepatitis B virus (HBV) replication in chronicallyinfected human hepatoblastoma 2.2.15 cells, with an effectiveconcentration (EC₅₀) of 0.25 μM, and a therapeutic index (IC₅₀C₅₀) of153. These values were compared with penciclovir and lamivudine (3TC),which exhibited EC₅₀ vlaues of 0.19 and 0.038 μM, respectively, andtherapeutic indexes of 471 and 11200. Combinations of robustaflavonewith penciclovir and lamivudine displayed synergistic anti-HBV activity,having the most pronounced effects when the combination ratios weresimilar to the ratio of EC₅₀ potencies. Thus, a 1:1 combination ofrobustaflavone and penciclovir exhibited an EC₅₀of 0.11 μM and atherapeutic index of 684, while a 10:1 combination of robustaflavone andlimivudine exhibited an EC₅₀ of 0.054 μM and a therapeutic index of 894.Measurement of extracellular and intracellular HBV DNA, HBV RNA andviral protein levels following exposure of 2.2.15 cells torobustaflavone indicated that only HBV DNA levels were affected,suggesting that inhibition of HBV DNA polymerase is the mechanism ofaction.

[0170] The results indicated that robustaflavone and robustaflavonetetrasulfate potassium salt were extremely effective anti-HBV agents.Robustaflavone also exhibited strong inhibitory effects againstinfluenza A and influenza B viruses. Both hinokiflavone androbustaflavone demonstrated similar activity against HIV-1 RT, producingIC₅₀ values of 35.2 μg/mL and 33.7 μg/mL, respectively. Amentoflavone,agathisflavone, morelloflavone, GB-1a and GB-2a were moderately activeagainst HIV-1 RT, with IC₅₀ values of 64.0 μg/mL, 53.8 μg/mL, 64.7μg/mL, 127.8 μg/mL, and 94.6 μg/mL, respectively. Morelloflavone alsodemonstrated significant antiviral activity against HIV-1 (strain LAV inphytohemagglutinin (PHA)-stimulated human peripheral blood mononuclear(PBM) cells) at an EC50 value of 5.7 μM and an SI value (selectivityindex) of approximately 10. The other biflavanoids were either slightlyactive or inactive against these viruses and HIV-1 RT.

[0171] Amentoflavanone (1), agathisflavone (2), volkensiflavanone (5),volkensiflavone hexamethyl ether (6), rhusflavanone (9), andsuccedaneaflavone (11) exhibited inhibitory activity against influenza Bvirus with the selective index (SI) of 178, 5.6, 34, ˜38, 9.3 and 15,respectively. Amentoflavone (1), and agathisflavone (2) alsodemonstrated anti-influenza A activity.

[0172] Robustaflavone (3) produced moderate inhibitory activity againstboth HSV-1 and HSV-2. Rhusflavanone (9) was active against HSV-2, whilesuccedaneaflavanone hexaacetate (12) was moderately active against VZV.

[0173] Comparison of robustaflavone with a series of other naturallyoccurring biflavanoids and biflavanones, as well as severalsemi-synthetic derivatives, indicated that robustaflavone prossessesunique structural features that impart the observed antiviral activity.The sermi-synthetic derivatives robustaflavone hexa-O-acetate androbustaflavone hexa-O-methyl ether were approximately three-fold and10-fold less potent with regard to anti-HBV activity, respectively, butneither of these derivatives exhibited cytotoxicity up to aconcentration of 1000 uM. Volkensiflavone hexa-O-methyl ether,rhusflavanone hexa-O-acetate and succedaneaflavanone hexa-O-acetate werethe only other non-robustaflavone analogues to inhibit HBV replication,but all possessed unacceptable selectivity indexes (1.3, 2.8 and 1.9,respectively). Interestingly, the parent compounds of the latter threehexa-O-acetate derivatives (volkensiflavone, rhusflavanone andsuccedaneaflavanone) were all inactive against HBV replication, incontrast to the relationship of robustaflavone with its hexaacetate.

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1. A method for treating or preventing a viral infection which comprisesadministering a composition comprising an antivirally effective amountof at least one biflavanoid and at least one antiviral agent.
 2. Themethod according to claim 1, wherein said biflavanoid is selected fromthe group consisting of robustaflavone, hinokiflavone, amentoflavone,agathisflavone, volkensiflavone, rhusflavanone, succedaneaflavanone,morelloflavone, GB-1a, GB-2a, derivatives or salts thereof.
 3. Themethod according to claim 2, wherein said biflavanoid is robustaflavone.4. The method according to claim 2, wherein said derivatives or saltsthereof comprise alkyl ethers, esters, acid adducts, amines andsulfates.
 5. The method according to claim 4, wherein saidrobustaflavone salt is robustaflavone tetrasulfate potassium salt. 6.The method according to claim 4, wherein said robustaflavone salt isrobustaflavone hexa-O-methyl ether.
 7. The method according to claim 4,wherein said robustaflavone salt is robustaflavone hexa-O-acetate. 8.The method according to claim 4, wherein said robustaflavone salt isrobustaflavone tetrasodium salt.
 9. The method according to claim 1,wherein said antiviral agent comprises AZT, ddC, ddI, D4T, lamivudine(3TC), alyclovir, gancyclovir, fluorinated nucleosides, TIBOderivatives, nevirapine, saquinavir, α-interferon and recombinant CD4),imnmunostimulants (e.g., various interleukins and cytokines),immunomodulators and antibiotics (e.g., antibacterial, antifungal,anti-pneumocysitis agents).
 10. The method according to claim 4, whereinsaid antiviral agent is 3TC.
 11. The method according to claim 1,wherein said biflavanoid is robustaflavone and said antiviral agent is3TC.
 12. The method according to claim 1, wherein said viral infectionis by an influenza virus.
 13. The method according to claim 12, whereinsaid influenza virus is influenza A or influenza B virus.
 14. The methodaccording to claim 1, wherein said viral infection is by a hepatitisvirus.
 15. The method according to claim 14, wherein said hepatitisvirus is hepatitis B virus.
 16. The method according to claim 1, whereinsaid viral infection is by a Herpes virus.
 17. The method according toclaim 16, wherein said Herpes virus is HSV-1 or HSV-2 virus.
 18. Themethod according to claim 1, wherein said viral infection is by aVaricella Zoster Virus (VZV).
 19. The method according to claim 18,wherein said viral infection is by a respiratory virus.
 20. The methodaccording to claim 19, wherein said respiratory virus is a measlesvirus.
 21. The method according to claim 1, wherein the viral infectionis by a retrovirus.
 22. The method according to claim 21, wherein theretrovirus is a human immunodeficiency virus (HIV).
 23. The methodaccording to claim 22, wherein human immunodeficiency virus (HIV) isHIV-
 1. 24. A pharmaceutical composition for treating and/or preventingviral infections which comprises an antivirally effective amount of atleast one substantially purified biflavanoid, at least one antiviralagent and a pharmaceutically acceptable carrier.
 25. The compositionaccording to claim 24 wherein said biflavanoid comprises robustaflavone,hinokiflavone, amentoflavone, agathisflavone, volkensiflavone,rhusflavanone, succedaneaflavanone, morelloflavone, GB-1a, GB-2a,derivatives or salts thereof.
 26. The composition according to claim 25,wherein said biflavanoid is robustaflavone.
 27. The compositionaccording to claim 25, wherein said derivatives or salts thereofcomprise alkyl ethers, esters, acid adducts, amines and sulfates. 28.The composition according to claim 27, wherein said derivatives or saltsthereof is a robustaflavone salt.
 29. The composition according to claim25, wherein said robustaflavone salt is robustaflavone tetrasulfatepotassium salt.
 30. The composition according to claim 25, wherein saidrobustaflavone salt is robustaflavone hexa-O-methyl ether.
 31. Thecomposition according to claim 25, wherein said robustaflavone isrobustaflavone hexa-O-acetate.
 32. The composition according to claim25, wherein said robustaflavone is robustaflavone tetrasodium salt. 33.The composition according to claim 24, wherein said antiviral agentcomprises AZT, ddC, ddI, D4T, lamivudine (3TC), alyclovir, gancyclovir,fluorinated nucleosides, TIBO derivatives, nevirapine, saquinavir,α-interferon and recombinant CD4), immunostimulants (e.g., variousinterleukins and cytokines), immunomodulators and antibiotics (e.g.,antibacterial, antifungal, anti-pneumocysitis agents).
 34. Thecomposition according to claim 33, wherein said antiviral agent is 3TC.35. The composition according to claim 24, wherein said biflavanoid isrobustaflavone and said antiviral agent is 3TC.
 36. A pharmaceuticalcomposition for treating and/or preventing viral infections whichcomprises an antivirally effective amount of a biflavanoid derivative.37. The composition of claim 36 wherein said biflavanoid derivativecomprises robustaflavone hexa-O-methyl ether, robustaflavonehexa-O-acetate, and robustaflavone tetrasodium salt and apharmaceutically acceptable carrier.
 38. A method for treating orpreventing a viral infection which comprises administering an antiviraleffective amount of a biflavanoid derivative.
 39. The method accordingto claim 38 wherein said biflavonoid derivative comprises robustaflavonehexa-O-methyl ether.
 40. The method according to claim 38 wherein saidbiflavonoid derivative comprises robustaflavone hexa-O-acetate.
 41. Themethod according to claim 38 wherein said biflavonoid derivativecomprises robustaflavone tetrasodium salt.
 42. A method for treating orpreventing a hepatitis B viral infection which comprises administering acomposition comprising an antivirally effective amount of robustaflavonehexa-O-acetate.
 43. A method for treating or preventing a hepatitis Bviral infection which comprises administering a composition comprisingan antivirally effective amount of robustaflavone hexa-O-methyl ether.44. A method for treating or preventing a hepatitis B viral infectionwhich comprises administering a composition comprising an antivirallyeffective amount of robustaflavone tetrasodium salt.
 45. The methodaccording to claims 42, 43, or 44, further comprising administering atleast one other antiviral agent.
 46. The method according to claim 45,wherein said antiviral agent comprises AZT, ddC, ddI, D4T, lamivudine(3TC), alyclovir, gancyclovir, fluorinated nucleosides, TIBOderivatives, nevirapine, saquinavir, α-interferon and recombinant CD4),immunostimulants (e.g., various interleukins and cytokines),immunomodulators and antibiotics (e.g., antibacterial, antifungal,anti-pneumocysitis agent).
 47. The method according to claim 45, whereinsaid antiviral agent comprises AZT, ddC, ddI, D4T, lamivudine (3TC),alyclovir, gancyclovir, fluorinated nucleosides, TIBO derivatives,nevirapine, saquinavir, α-interferon and recombinant CD4),immunostimulants (e.g., various interleukins and cytokines),immunomodulators and antibiotics (e.g., antibacterial, antifungal,anti-pneumocysitis agents).
 48. The method according to claims 45,wherein said antiviral agent is 3TC.
 49. A pharmaceutical compositionfor treating and/or preventing a hepatitis B viral infection whichcomprises an antivirally effective amount of robustaflavonehexa-O-acetate and a pharmaceutically acceptable carrier.
 50. Apharmaceutical composition for treating and/or preventing a hepatitis Bviral infection which comprises an antivirallv effective amount ofrobustaflavone hexa-O-methyl ether and a pharmaceutically acceptablecarrier.
 51. A pharmaceutical composition for treating and/or preventinga hepatitis B viral infection which comprises an antivirally effectiveamount of robustaflavone tetradsodium salt and a pharmaceuticallyacceptable carrier.
 52. The pharmaceutical composition according toclaims 49-51 further comprising at least one other anti-viral agentcomprising AZT, ddC, ddI, D4T, lamivudine (3TC), alyclovir, gancyclovir,fluorinated nucleosides, TIBO derivatives, nevirapine, saquinavir,α-interferon and recombinant CD4), immunostimulants (e.g., variousinterleukins and cytokines), immunomodulators and antibiotics (e.g.,antibacterial, antifungal, anti-pneumocysitis agents).